Methods for quantitative determination of methylation density in a dna locus

ABSTRACT

The present invention is a novel method of determining the average DNA methylation density of a locus of interest within a population of DNA fragments.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/971,986, filed Oct. 21, 2004, which claims benefit ofpriority to U.S. Provisional Patent Application No. 60/561,721, filedApr. 12, 2004, U.S. Provisional Patent Application No. 60/561,563, filedApr. 12, 2004, and U.S. Provisional Patent Application No. 60/513,426,filed Oct. 21, 2003, each of which are incorporated by reference intheir entirety for all purposes.

BACKGROUND OF THE INVENTION

Human cancer cells typically contain somatically altered genomes,characterized by mutation, amplification, or deletion of critical genes.In addition, the DNA template from human cancer cells often displayssomatic changes in DNA methylation. See, e.g., E. R. Fearon, et al, Cell61:759 (1990); P. A. Jones, et al., Cancer Res. 46:461 (1986); R.Holliday, Science 238:163 (1987); A. De Bustros, et al., Proc. Natl.Acad. Sci. USA 85:5693 (1988); P. A. Jones, et al., Adv. Cancer Res.54:1 (1990); S. B. Baylin, et al., Cancer Cells 3:383 (1991); M. Makos,et al., Proc. Natl. Acad Sci. USA 89:1929 (1992); N. Ohtani-Fujita, etal., Oncogene 8:1063 (1993).

DNA methylates transfer methyl groups from the universal methyl donorS-adenosyl methionine to specific sites on the DNA. Several biologicalfunctions have been attributed to the methylated bases in DNA. The mostestablished biological function is the protection of the DNA fromdigestion by cognate restriction enzymes. This restriction modificationphenomenon has, so far, been observed only in bacteria.

Mammalian cells, however, possess a different methylase that exclusivelymethylates cytosine residues on the DNA that are 5′ neighbors of guanine(CpG). This methylation has been shown by several lines of evidence toplay a role in gene activity, cell differentiation, tumorigenesis,X-chromosome inactivation, genomic imprinting and other major biologicalprocesses (Razin, A., H., and Riggs, R. D. eds. in DNA MethylationBiochemistry and Biological Significance, Springer-Verlag, N.Y., 1984).

In eukaryotic cells, methylation of cytosine residues that areimmediately 5′ to a guanosine, occurs predominantly in CG poor loci(Bird, A., Nature 321:209 (1986)). In contrast, discrete regions of CGdinucleotides called CpG islands remain unmethylated in normal cells,except during X-chromosome inactivation and parental specific imprinting(Li, et al., Nature 366:362 (1993)) where methylation of 5′ regulatoryregions can lead to transcriptional repression. For example, de novomethylation of the Rb gene has been demonstrated in a small fraction ofretinoblastomas (Sakai, et al., Am. J. Hum. Genet., 48:880 (1991)), anda more detailed analysis of the VHL gene showed aberrant methylation ina subset of sporadic renal cell carcinomas (Herman, et al., Proc. Natl.Acad. Sci. U.S.A., 91:9700 (1994)). Expression of a tumor suppressorgene can also be abolished by de novo DNA methylation of a normallyunmethylated 5′ CpG island. See, e.g., Issa, et al., Nature Genet. 7:536(1994); Merlo, et al., Nature Med. 1:686 (1995); Herman, et al., CancerRes., 56:722 (1996); Graff, et al., Cancer Res., 55:5195 (1995); Herman,et al., Cancer Res. 55:4525 (1995).

Identification of the earliest genetic changes in tumorigenesis is amajor focus in molecular cancer research. Diagnostic approaches based onidentification of these changes can allow implementation of earlydetection strategies, tumor staging and novel therapeutic approachestargeting these early changes, leading to more effective cancertreatment. The present invention addresses these and other problems.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for quantifying the averagemethylation density in a target sequence within a population of genomicDNA. In some embodiments, the method comprises contacting genomic DNAwith a methylation-dependent restriction enzyme or methylation-sensitiverestriction enzyme under conditions that allow for at least some copiesof potential restriction enzyme cleavage sites in the locus to remainuncleaved; quantifying intact copies of the locus; and comparing thequantity of amplified product to a control value representing thequantity of methylation of control DNA, thereby quantifying the averagemethylation density in the locus compared to the methylation density ofthe control DNA.

In some embodiments, the quantifying step comprises a quantitativeamplification. In some embodiments, the quantity of the amplifiedproduct is compared to a standard curve.

In some embodiments, the quantifying step comprises the direct detectionof intact copies of locus with hybrid capture.

In some embodiments, the amplifying step comprises hybridizing twooligonucleotide primers to DNA flanking the locus to produce anamplification product corresponding to the uncleaved locus of genomicDNA between the primers.

In some embodiments, the control value represents the quantity of anamplification product of a DNA sample having a known or predicted numberof methylated nucleotides.

In some embodiments, the restriction enzyme is a methylation-sensitiverestriction enzyme. In some embodiments, the methylation-sensitiverestriction enzyme is selected from the group consisting of Aat II, AciI, Acl I, Age I, Alu I, Asc I, Ase I, AsiS I, Bbe I, BsaA I, BsaH I,BsiE I, BsiW I, BsrF I, BssH II, BssK I, BstB I, BstN I, BstU I, ClaIEae I, Eag I, Fau I, Fse I, Hha I, HinP1 I, HinC II, Hpa II, Hpy99 I,HpyCH4 IV, Kas I, Mlu I, MapA1 I, Msp I, Nae I, Nar I, Not I, Pml I, PstI, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, Sfl I, Sfo I, SgrA I, Sma I,SnaB I, Tsc I, Xma I, and Zra I.

In some embodiments, the restriction enzyme is a methylation-dependentrestriction enzyme. In some embodiments, the restriction enzyme is amethyl-cytosine-dependent restriction enzyme. In some embodiments, therestriction enzyme is McrBC. In some embodiments, the restriction enzymeis a methyl-adenosine-dependent restriction enzyme. In some embodiments,the restriction enzyme is DpnI.

In some embodiments, the methylation-sensitive or methylation dependentrestriction enzyme is contacted to the portion under conditions to allowfor only a partial digest of the portion.

In some embodiments, the method comprises separating the genomic DNAinto at least two equal portions; contacting one portion with amethylation-sensitive or methylation dependent restriction enzyme andcontacting a second portion with the isoschizomeric partner of therestriction enzyme, amplifying the locus of genomic DNA in each portionin a step comprising hybridizing two oligonucleotide primers to DNAflanking the locus; quantifying the amplification product; and comparingthe quantity of amplified products from the two portions.

In some embodiments, the method further comprises contacting the genomicDNA with an agent that modifies unmethylated cytosine before theamplifying step, and at least one of the two oligonucleotide primersdistinguishes between modified unmethylated and methylated DNA in thegenomic DNA.

In some embodiments, the method further comprises contacting the DNAwith at least one methylation-sensitive restriction enzyme ormethylation-dependent restriction enzyme before the genomic DNA iscontacted with an agent that modifies unmethylated cytosine. In someembodiments, the genomic DNA is contacted with a mixture of at least twodifferent methylation-dependent or methylation-sensitive restrictionenzymes.

In some embodiments, the agent is sodium bisulfite.

In some embodiments, the amplified product is quantified usingquantitative PCR.

In some embodiments, the control value is generated by contacting DNAcomprising a control locus with a methylation-dependent ormethylation-sensitive restriction enzyme; amplifying the control locus;and determining the quantity of the amplified product. In someembodiments, the control locus is known or predicted to be unmethylated.

In some embodiments, the control value comprises a known number ofmethylated nucleotides. In some embodiments, the genomic DNA is from ahuman. In some embodiments, the method is performed to detect thepresence or absence of cancer cells in a subject.

In some embodiments, the quantifying step comprises detecting a probethat hybridizes to the amplification product. In some embodiments, theprobe comprises a detectable fluorescent moiety.

In some embodiments, the quantifying step comprises the direct detectionof intact copies of locus with hybrid capture.

In some embodiments, the DNA is from an animal. In some embodiments, theanimal is a human.

In some embodiments, the genomic DNA is from a tissue selected from thegroup consisting of brain tissue, colon tissue, urogenital tissue, lungtissue, renal tissue, hematopoietic tissue, breast tissue, thymustissue, testis tissue, ovarian tissue, uterine tissue and blood.

In some embodiments, the genomic DNA is from an organism selected fromthe group consisting of plants, fungi and bacteria.

The present invention also provides methods of calculating the relativemethylation density for a target locus in a DNA sample. In someembodiments, the methods comprise

i. contacting the DNA sample with a methylation-dependent restrictionenzyme under conditions that allow for at least some copies of potentialrestriction enzyme cleavage sites in the locus to remain uncleaved toobtain a population of nucleic acids in which at least some methylatedcopies of the locus are fragmented, or

contacting the DNA sample with a methylation-sensitive restrictionenzyme under conditions that allow for at least some copies of potentialrestriction enzyme cleavage sites in the locus to remain uncleaved toobtain a population of nucleic acids in which at least some unmethylatedcopies of the locus are fragmented;

ii. quantifying the number of intact copies of the locus in the DNAusing hybrid capture; andiii. determining the relative methylation density for the locus bycomparing the hybrid capture signal of a portion of a sample to thehybrid capture signal of a different portion of the sample or to acontrol value (as described herein).

The present invention also provides methods of calculating the relativemethylation density for a target locus in a DNA sample. In someembodiments, the methods comprise

i. contacting the DNA sample with a methylation-dependent restrictionenzyme under conditions that allow for at least some copies of potentialrestriction enzyme cleavage sites in the locus to remain uncleaved toobtain a population of nucleic acids in which at least some methylatedcopies of the locus are fragmented, or

contacting the DNA sample with a methylation-sensitive restrictionenzyme under conditions that allow for at least some copies of potentialrestriction enzyme cleavage sites in the locus to remain uncleaved toobtain a population of nucleic acids in which at least some unmethylatedcopies of the locus are fragmented;

ii. quantitatively amplifying intact copies of the locus in the DNAsample after the contacting steps;iii. identifying the cycle threshold (Ct) value for the amplifiedportion from the DNA sample; and,iv. determining the relative methylation density for the target locus bycalculating the difference (ΔCt) between the Ct of the DNA sample and acontrol Ct value, wherein 2^(|ΔCt|) equals, or is proportional to therelative methylation density between the DNA sample and the control.

In some embodiments, the control Ct is calculated by steps comprising

i. contacting a control DNA sample with a methylation-dependentrestriction enzyme under conditions that allow for at least some copiesof potential restriction enzyme cleavage sites in the locus to remainuncleaved to obtain a population of nucleic acids in which at least somemethylated copies of the locus are fragmented, or

contacting the control DNA sample with a methylation-sensitiverestriction enzyme under conditions that allow for at least some copiesof potential restriction enzyme cleavage sites in the locus to remainuncleaved to obtain a population of nucleic acids in which at least someunmethylated copies of the locus are fragmented;

ii. amplifying intact copies of the locus in the control DNA sampleafter the contacting steps; and,iii. identifying the cycle threshold (Ct) value for the amplifiedportion from the control DNA sample.

In some embodiments, the amplifying step comprises hybridizing twooligonucleotide primers to DNA flanking the locus to produce anamplification product corresponding to the uncleaved locus of genomicDNA between the primers. In some embodiments, the restriction enzyme isa methylation-sensitive restriction enzyme. In some embodiments, themethylation-sensitive restriction enzyme is selected from the groupconsisting of Aat II, Aci I, Acl I, Age I, Alu I, Asc I, Ase I, AsiS I,Bbe I, BsaA I, BsaH I, BsiE I, BsiW I, BsrF I, BssH II, BssK I, BstB I,BstN I, BstU I, ClaI, Eae I, Eag I, Fau I, Fse I, Hha I, HinP1 I, HinCII, Hpa II, Hpy99 I, HpyCH4 IV, Kas I, Mlu I, MapA1 I, Msp I, Nae I, NarI, Not I, Pml I, Pst I, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, Sfl I,Sfo I, SgrA I, Sma I, SnaB I, Tsc I, Xma I, and Zra I.

In some embodiments, the methylation-sensitive restriction enzyme doesnot cut when an adenosine within the recognition sequence is methylatedat position N6. In some embodiments, the methylation-sensitiverestriction enzyme is Mbo I.

In some embodiments, the restriction enzyme is a methylation-dependentrestriction enzyme. In some embodiments, the restriction enzyme is amethyl-cytosine-dependent restriction enzyme. In some embodiments, therestriction enzyme is McrBC, McrA, and MrrA. In some embodiments, therestriction enzyme is a methyl-adenosine-dependent restriction enzyme.In some embodiments, the restriction enzyme is DpnI.

In some embodiments, the methylation-sensitive or methylation dependentrestriction enzyme is contacted to the portion under conditions to allowfor only a partial digest of the portion.

The present invention also provides kits for quantifying the averagemethylation density in a locus of genomic DNA. In some embodiments, thekit comprises a methylation-dependent restriction enzyme or amethylation sensitive restriction enzyme; a control DNA moleculecomprising a pre-determined number of methylated nucleotides; andcontrol oligonucleotide primers that hybridize to the control DNAmolecule.

In some embodiments, the restriction enzyme is a methylation-sensitiverestriction enzyme. In some embodiments, the restriction enzyme is amethylation-dependent restriction enzyme. In some embodiments, therestriction enzyme is a methyl-cytosine-dependent restriction enzyme. Insome embodiments, the restriction enzyme is McrBC.

In some embodiments, the kit further comprises target oligonucleotideprimers that hybridize to a pre-determined locus of human genomic DNA.In some embodiments, at least one target oligonucleotide primerdistinguishes between modified unmethylated and methylated DNA in humangenomic DNA. In some embodiments, the kit comprises a plurality of DNAmolecules comprising different pre-determined numbers of methylatednucleotides. In some embodiments, the kit further comprises reagentssufficient to support the activity of the restriction enzyme. In someembodiments, the kit further comprises a thermostable DNA polymerase. Insome embodiments, the kit further comprises an agent that modifiesunmethylated cytosine. In some embodiments, the kit further comprises adetectably-labeled oligonucleotide. In some embodiments, the kitcomprises hybrid capture reagents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate results of amplification of DNA at differentmethylated:unmethylated dilutions.

FIG. 2 illustrates the ability of McrBC to distinguish between DNA atdifferent methylated:unmethylated dilutions. The arrows at the bottom ofthe figure indicate the approximate ΔCt between HhaI-cut and HhaI/McrBCdouble cut samples.

FIG. 3 illustrates analysis of DNA at a 1:2000 methylated:unmethylateddilution.

FIG. 4 illustrates a plot of change in cycle threshold as a function ofdilution of methylated/unmethylated DNA.

FIG. 5 illustrates results from different methylated:unmethylateddilutions.

FIG. 6 illustrates a hypothetical methylation density progression in thedevelopment of disease.

FIG. 7 illustrates McrBC DNA restriction.

FIG. 8 illustrates amplification results from different McrBC dilutionsrestricting sparsely-methylated DNA.

FIG. 9 illustrates amplification results from different McrBC dilutionsrestricting densely-methylated DNA.

FIG. 10 illustrates using different restriction enzyme dilutions todetermine optimum resolution between DNA with different methylationdensities.

FIG. 11 illustrates what data is obtained when the methylation state ofonly particular nucleotides is detected in a hypothetical diseaseprogression.

FIG. 12 illustrates what data is obtained when the average methylationdensity of a locus is detected in a hypothetical disease progression.

FIG. 13 illustrates comparison of different restriction enzyme digeststo provide additional analysis of DNA methylation.

FIG. 14 illustrates analysis of McrBC/amplification-based methylationdetection and comparison to bisulfite sequencing. The data was generatedusing bisulfite treatment, McrBC digestion, and then amplification.

FIG. 15 depicts a portion of the p16 promoter (SEQ ID NO:1) methylatedin vitro with M.Sss I.

FIG. 16 illustrates data demonstrating that methylation-dependent (i.e.,McrBC) and methylation-sensitive (i.e., Aci I) restriction enzymesdistinguish different methylation densities at a DNA locus.

FIG. 17 illustrates cycle threshold data demonstrating thatmethylation-dependent (i.e., McrBC) and methylation-sensitive (i.e., AciI) restriction enzymes distinguish different methylation densities at aDNA locus.

FIG. 18 illustrates a consensus restriction map of kafirin genes. Therelevant restriction sites are indicated vertically and the numbersindicate the distances scale in base-pairs. Each coding sequence isdepicted as the blue-shaded arrow, and the region assayed is indicatedby the black bar. The circles depict sites that are not present in everykafirin gene, and the color represents the number of genes that do notshare the site. The orange circle (5′ most HhaI site) is conserved in 9of 11 Kafirin genes, and the red circle (3′ most PstI site) is presentin 10 of the 11.

FIG. 19 illustrates the heterogenous CG methylation and homogenous CNGmethylation of eleven kafirin genes.

DEFINITIONS

A “fragment” of DNA refers to an intact DNA molecule of variable size,which can be an entire chromosome or smaller segments thereof

“Methylation” refers to methylation at positions C⁵ or N⁴ of cytosine,the N⁶ position of adenosine or other types of nucleic acid methylation.

A “methylation-dependent restriction enzyme” refers to a restrictionenzyme that cleaves at or near a methylated recognition sequence, butdoes not cleave at or near the same sequence when the recognitionsequence is not methylated. Methylation-dependent restriction enzymescan recognize, for example, specific sequences comprising amethylated-cytosine or a methylated-adenosine. Methylation-dependentrestriction enzymes include those that cut at a methylated recognitionsequence (e.g., DpnI) and enzymes that cut at a sequence that is not atthe recognition sequence (e.g., McrBC). For example, McrBC requires twohalf-sites. Each half-site must be a purine followed by5-methyl-cytosine (R5mC) and the two half-sites must be no closer than20 base pairs and no farther than 4000 base pairs away from each other(N20-4000). McrBC generally cuts close to one half-site or the other,but cleavage positions are typically distributed over several base pairsapproximately 32 base pairs from the methylated base. Exemplarymethylation-dependent restriction enzymes include, e.g., McrBC (see,e.g., U.S. Pat. No. 5,405,760), McrA, MrrA, and Dpn I. One of skill inthe art will appreciate that homologs and orthologs of the restrictionenzymes described herein are also suitable for use in the presentinvention.

A “methylation insensitive restriction enzyme” refers to a restrictionenzyme that cuts DNA regardless of the methylation state of the base ofinterest (A or C) at or near the recognition sequence.

A “methylation sensing restriction enzyme” refers to a restrictionenzyme whose activity changes in response to the methylation of itsrecognition sequence.

A “methylation-sensitive restriction enzyme” refers to a restrictionenzyme (e.g., PstI) that cleaves at or in proximity to an unmethylatedrecognition sequence but does not cleave at or in proximity to the samesequence when the recognition sequence is methylated. Exemplary5′-methyl cytosine sensitive restriction enzymes include, e.g., Aat II,Aci I, Acl I, Age I, Alu I, Asc I, Ase I, AsiS I, Bbe I, BsaA I, BsaH I,BsiE I, BsiW I, BsrF I, BssH II, BssK I, BstB I, BstN I, BstU I, Cla I,Eae I, Eag I, Fau I, Fse I, Hha I, HinP1 I, HinC II, Hpa II, Hpy99 I,HpyCH4 IV, Kas I, Mlu I, MapA1 I, Msp I, Nae I, Nar I, Not I, Pml I, PstI, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, Sfl I, Sfo I, SgrA I, Sma I,SnaB I, Tsc I, Xma I, or Zra I. See e.g., McClelland, M. et al, NucleicAcids Res. 22(17):3640-59 (1994) and http://rebase.neb.com. Exemplarymethyl adenosine sensitive restriction enzymes include, e.g., MboI.

As used herein, a “recognition sequence” refers only to a primarynucleic acid sequence and does not reflect the methylation status of thesequence.

The “methylation density” refers to the number of methylated residues ina given locus of DNA divided by the total number of nucleotides in thesame DNA sequence that are capable of being methylated. Methylationdensity may be determined for methylated-cytosines ormethylated-adenosines.

Cleaving DNA “under conditions that allow for at least some copies ofpotential restriction enzyme cleavage sites in the locus to remainuncleaved” refers to any combination of reaction conditions, restrictionenzyme and enzyme concentration and/or DNA resulting in at least some ofthe DNA comprising a potential restriction enzyme cleavage site toremain uncut. For example, a partial digestion of the DNA (e.g., bylimiting the amount of enzyme or the amount of time of the digestion)allows some potential restriction enzyme cleavage sites in the locus toremain uncut. Alternatively, a complete digestion using a restrictionenzyme such as McrBC will result in some potential restriction enzymecleavage sites in the locus to remain uncut because the enzyme does notalways cut between the two recognition half sites, thereby leaving atleast some uncleaved copies of a locus in a population of sequenceswherein the locus is defined by the two recognition half-sites. A“potential restriction enzyme cleavage site” refers to a sequence that arestriction enzyme is capable of cleaving (i.e., comprising theappropriate nucleotide sequence and methylation status) when itrecognizes the enzymes recognition sequence, which may be the same ordifferent from the cleavage site.

“Amplifying” DNA refers to any chemical, including enzymatic, reactionthat results in an increased number of copies of a template nucleic acidsequence. Amplification reactions include polymerase chain reaction(PCR) and ligase chain reaction (LCR) (see U.S. Pat. Nos. 4,683,195 and4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis etal., eds, 1990)), strand displacement amplification (SDA) (Walker, etal. Nucleic Acids Res. 20(7):1691-6 (1992); Walker PCR Methods Appl3(1):1-6 (1993)), transcription-mediated amplification (Phyffer, et al.,J. Clin. Microbiol. 34:834-841 (1996); Vuorinen, et al., J. Clin.Microbiol. 33:1856-1859 (1995)), nucleic acid sequence-basedamplification (NASBA) (Compton, Nature 350(6313):91-2 (1991), rollingcircle amplification (RCA) (Lisby, Mol. Biotechnol. 12(1):75-99 (1999));Hatch et al., Genet. Anal. 15(2):35-40 (1999)); branched DNA signalamplification (bDNA) (see, e.g., Iqbal et al., Mol. Cell Probes13(4):315-320 (1999)); and linear amplification.

A “partial digestion” of DNA as used herein refers to contacting DNAwith a restriction enzyme under appropriate reaction conditions suchthat the restriction enzyme cleaves some (e.g., less than about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) but not all of possiblecleavage sites for that particular restriction enzyme in the DNA. Apartial digestion of the sequence can be achieved, e.g., by contactingDNA with an active restriction enzyme for a shorter period of time thanis necessary to achieve a complete digestion and then terminating thereaction, or under other altered reaction conditions that allow for thedesired amount of partial digestion. “Possible sites” are generallyenzyme recognition sequences, but also include situations where anenzyme cleaves at a sequence other than the recognition sequence (e.g.,McrBC).

A “complete digestion” of DNA as used herein refers to contacting DNAwith a restriction enzyme for sufficient time and under appropriateconditions to allow for cleavage of at least 95%, and typically at least99%, or all of the restriction enzyme recognition sequences for theparticular restriction enzyme. Conditions, including the time, buffersand other reagents necessary for complete digestions are typicallyprovided by manufacturers of restriction enzymes. Those of skill in theart will recognize that the quality of the DNA sample may preventcomplete digestion.

“Isoschizomers” refer to restriction enzymes that recognize the samenucleotide sequence. As used in this definition, the “same nucleotidesequence” is not intended to differentiate between methylated andunmethylated sequences. Thus, an “isoschizomeric partner” of amethylation-dependent or methylation-sensitive restriction enzyme is arestriction enzyme that recognizes the same recognition sequence as themethylation-dependent or methylation-sensitive restriction enzymeregardless of whether the recognition sequence is methylated.

“An agent that modifies unmethylated cytosine” refers to any agent thatalters the chemical composition of unmethylated cytosine but does notchange the chemical composition of methylated cytosine. An example ofsuch an agent is sodium bisulfite.

“Primers that distinguish between methylated and unmethylated DNA”refers to oligonucleotides that:

-   -   (i) hybridize (e.g., are at least partially complementary) to a        sequence that represents a methylated DNA sequence after        bisulfite conversion, but do not hybridize to a sequence        representing the identical unmethylated sequence after bisulfite        conversion; or    -   (ii) hybridize to a sequence that represents an unmethylated DNA        sequence after bisulfite conversion, but do not hybridize to a        sequence representing the identical methylated sequence after        bisulfite conversion.

As described herein, primers that distinguish between methylated andunmethylated sequences are generally designed to hybridize to a sequencethat would occur if the DNA was treated with an agent (such as sodiumbisulfite) that modifies unmethylated nucleotides but not methylatednucleotides or vice versa. For example, when sodium bisulfite iscontacted to DNA, unmethylated cytosine is converted to uracil, whilemethylated cytosine is not modified. Since uracil forms complements withadenine, a primer that binds to the unmethylated sequence would containadenines at locations, where the adenines would form complements withthe modified cytosines (i.e., uracils). Similarly, if a primer thathybridized to sequences containing methylated cytosines was desired, theprimer would contain guanosines, where it would form complements withthe methylated cytosines. Thus, sequences that “represent” methylated orunmethylated DNA include DNA that result from sodium bisulfite treatmentof the DNA.

A “locus” as used herein refers to a target sequence within a populationof nucleic acids (e.g., a genome). If a single copy of the targetsequence is present in the genome, then “locus” will refer to a singlelocus. If multiple copies of the target sequence are present in thegenome, then “locus” will refer to all loci that contain the targetsequence in the genome.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention provides rapid and efficient methods fordetermining the presence of methylation and the methylation density inregions of genomic DNA. Determination of alterations in methylationdensity can be useful for providing diagnoses and prognoses for variousdiseases, including various cancers. While the methods of the inventionalso provide for the detection of specific methylation events, thepresent methods are particularly notable because they are not limited bya prediction or expectation that the methylation state of a particularnucleotide is determinative of a phenotype. In cases where the densityof methylation (i.e., the quantity of nucleotides that are methylated ina particular length of a nucleic acid sequence), rather than thepresence or absence of a particular methylated nucleotide, modulatesgene expression, and where the methylation density of a locus reflectsdisease progression along a continuum, the present methods areparticularly helpful.

II. Quantifying the Relative Amount of Methylation in Genomic DNA

The quantity of methylation of a locus of DNA can be determined byproviding a sample of genomic DNA comprising the locus, cleaving the DNAwith a restriction enzyme that is either methylation-sensitive ormethylation-dependent, and then quantifying the amount of intact DNA orquantifying the amount of cut DNA at the DNA locus of interest. Theamount of intact or cut DNA will depend on the initial amount of genomicDNA containing the locus, the amount of methylation in the locus, andthe number (i.e., the fraction) of nucleotides in the locus that aremethylated in the genomic DNA. The amount of methylation in a DNA locuscan be determined by comparing the quantity of intact DNA or cut DNA toa control value representing the quantity of intact DNA or cut DNA in asimilarly-treated DNA sample. As discussed below, the control value canrepresent a known or predicted number of methylated nucleotides.Alternatively, the control value can represent the quantity of intact orcut DNA from the same locus in another (e.g., normal, non-diseased) cellor a second locus.

As discussed in detail below, by using at least onemethylation-sensitive or methylation-dependent restriction enzyme underconditions that allow for at least some copies of potential restrictionenzyme cleavage sites in the locus to remain uncleaved and subsequentlyquantifying the remaining intact copies and comparing the quantity to acontrol, average methylation density of a locus may be determined. Ifthe methylation-sensitive restriction enzyme is contacted to copies of aDNA locus under conditions that allow for at least some copies ofpotential restriction enzyme cleavage sites in the locus to remainuncleaved, then the remaining intact DNA will be directly proportionalto the methylation density, and thus may be compared to a control todetermine the relative methylation density of the locus in the sample.Similarly, if a methylation-dependent restriction enzyme is contacted tocopies of a DNA locus under conditions that allow for at least somecopies of potential restriction enzyme cleavage sites in the locus toremain uncleaved, then the remaining intact DNA will be inverselyproportional to the methylation density, and thus may be compared to acontrol to determine the relative methylation density of the locus inthe sample.

A. Digestion with Restriction Enzymes

Either partial or complete restriction enzyme digestions can be used toprovide information regarding methylation density within a particularDNA locus.

i. Complete Digestion

When a DNA sample comprising a locus of interest is completely digestedwith a methylation sensing restriction enzyme, the information providedincludes the presence or absence of methylation at recognition sequencesof the restriction enzyme. The presence of intact DNA in a locuscomprising the cut site of the restriction enzyme indicates that theappropriate methylation state of the recognition site necessary forcleavage by the methylation-sensitive or methylation-dependentrestriction enzyme was not present at or near the locus, depending onthe restriction enzyme.

The amount of intact test DNA can be compared to a control representingan equal amount of DNA from the sample that was not contacted with therestriction enzyme. Alternatively, the amount of intact DNA at a locuscan be compared to similarly-treated intact DNA comprising a secondlocus or compared to the same locus in DNA isolated from another cellwhen all DNA samples are treated similarly. In another alternative, theamount of intact DNA at a locus can be compared to similarly-treated DNAhaving a known or expected number of methylated and monitorablerestriction sites and comparable in size. Those of skill in the art willappreciate that other controls are also possible. Thus, by detecting theamount of intact DNA at the locus following restriction enzymedigestion, the relative number of methylated copies compared to thetotal number of copies of the locus is determined.

Use of restriction enzymes that have a variable cleavage pattern nearthe recognition sequence (e.g., McrBC) provides a special case forcomplete digestions of DNA. In this case, even if the locus contains arecognition sequence in the appropriate methylation state, some of thefragments containing a methylated locus will remain intact becausecleavage of the DNA will occur outside the locus according to a functionof probability. Therefore, a complete digestion with McrBC behavessimilarly to a partial digestion with a methylation sensing restrictionenzyme (which cuts at its recognition site) with respect to the numberof intact alleles.

The mechanism of McrBC DNA cutting occurs as follows. An eight subunitcomplex of McrB binds to each of two recognition half sites(purine-methylC represented as (A or G)mC). See FIG. 7. These complexesthen recruit one McrC subunit to their respective half sites and startto translocate along the DNA mediated by GTP hydrolysis. When two McrBCbound complexes contact each other, a double-complex is formed andrestriction occurs. Cutting will generally not occur if the two halfsites are closer than 20 bp and restriction resulting from half sites asfar as 4 kb from one another have been observed, though are rare.Restriction may occur ˜32 bp to the left or right of either bound halfsite, giving four possible cut site locations: ˜32 bp 5′ of the firsthalf site, ˜32 bp 3′ of the first half site, ˜32 bp 5′ of the secondhalf site, and ˜32 bp 3′ of the second half site. Therefore, it ispossible for two half sites to exist within a locus defined by PCRprimers and for cleavage to occur outside of the locus. It is alsopossible for two half sites to exist outside of the locus and for a cutto occur within the locus. It is also possible for one site to exist inthe locus and for another to exist outside of the locus and for a cut tooccur either within or outside of the locus. Thus, the more methylatedhalf sites that are “in the vicinity” of the locus (whether literallybetween the amplification primers or in neighboring flanking sequence),the more likely a cut will be observed within the locus for a givenconcentration of McrBC. Accordingly, the number of copies of amethylated locus that are cleaved by McrBC in a complete or partialdigestion will be proportional to the density of methylated nucleotides.

ii. Partial Digestions

The amount of cleavage with a methylation sensitive ormethylation-dependent restriction enzyme in a partial (i.e., incomplete)digestion reflects the average methylation density within the locus ofDNA in the sample. For instance, when a locus has a higher methylationdensity than a control, then a partial digestion using amethylation-dependent restriction enzyme will cleave copies of the locusmore frequently. Similarly, when a locus has a lower methylation densitythan a control, then a partial digestion using a methylation-dependentrestriction enzyme will cleave copies of the locus less frequentlywithin the locus because fewer recognition sites are present.Alternatively, when a methylation sensitive restriction enzyme is used,fewer copies of a locus with a higher methylated density are cleavedless, and thus more intact DNA strands containing the locus are present.In each of these cases, the digestion of DNA sample in question iscompared to a control value such as those discussed above for completedigestions. Alternatively, the quantity of intact DNA after digestioncan be compared to a second sample to determine relative methylationdensity between the samples.

It can be useful to test a variety of conditions (e.g., time ofrestriction, enzyme concentration, different buffers or other conditionsthat affect restriction) to identify the optimum set of conditions toresolve subtle or gross differences in methylation density among two ormore samples. The conditions may be determined for each sample analyzedor may be determined initially and then the same conditions may beapplied to a number of different samples.

iii. DNA Samples

DNA can be obtained from any biological sample can be used, e.g., fromcells, tissues, secretions, and/or fluids from an organism (e.g., ananimal, plant, fungus, or prokaryote). The samples may be fresh, frozen,preserved in fixative (e.g., alcohol, formaldehyde, paraffin, orPreServeCyte™) or diluted in a buffer. Biological samples include, e.g.,skin, blood or a fraction thereof, tissues, biopsies (from e.g., lung,colon, breast, prostate, cervix, liver, kidney, brain, stomach,esophagus, uterus, testicle, skin, hair, bone, kidney, heart, gallbladder, bladder, and the like), body fluids and secretions (e.g.,blood, urine, mucus, sputum, saliva, cervical smear specimens, marrow,feces, sweat, condensed breath, and the like). Biological samples alsoinclude, leaves, stems, roots, seeds, petals, pollen, spore, mushroomcaps, and sap.

The above-described digestions can be used to analyze a sample of DNAwhere all copies of a genomic DNA locus have an identical methylationpattern. In other embodiments, the DNA sample is a mixture of DNAcomprising alleles of a DNA locus in which some alleles are moremethylated than others. In some embodiments, a DNA sample contains DNAfrom two or more different cell types, wherein each cell type has adifferent methylation density at a particular locus. For example, atsome loci, neoplastic cells have different methylation densitiescompared to normal cells. If a tissue, body fluid, or secretion containsDNA from both normal and neoplastic cells, then the DNA sample from thetissue, body fluid, or secretion will comprise a heterogeneous mixtureof differentially methylated alleles. In this case, at a given locus,one set of alleles within the DNA (e.g., those derived from neoplasticcells in the sample) will have a different methylation density than theother set of alleles (e.g., those derived from normal cells).

In mixed samples (e.g., in biopsies comprising healthy and diseasedcell), it may be helpful to focus results on one population of nucleicacids in the sample (e.g., from diseased cells) rather than to determinethe average methylation density across DNA from all cells in the sample.In some embodiments in which a first population of DNA in the sample haslow or no methylation and the second population of DNA in the sample hasmore methylation than the first population, density in the secondpopulation can be determined by cleaving the sample with one or moremethylation-sensitive restriction enzymes (generally cut to“completion”), thereby degrading the first population while leaving thesecond population substantially intact. Thus, the sample may also becontacted with a methylation-dependent restriction enzyme (using McrBCand/or any methylation-dependent restriction enzyme under partialdigestion conditions) and the remaining intact DNA may be amplified,thereby determining the methylation density in the second population.The methylation density of the first population may be similarlydetermined by contacting the sample with one or moremethylation-dependent restriction enzymes (generally cut to“completion”) and contacting the sample with a methylation sensitiveunder partial digestion conditions. In this case, the amplified DNA willrepresent the methylation density of the first population.

B. Amplification to Detect Intact DNA

The presence and quantity of DNA cleaved by the restriction enzymes canbe determined by amplifying the locus following digestion. By usingamplification techniques (e.g., the polymerase chain reaction (PCR))that require the presence of an intact DNA strand for amplification, thepresence and amount of remaining uncut DNA can be determined. Forexample, PCR reactions can be designed in which the amplificationprimers flank a particular locus of interest. Amplification occurs whenthe locus comprising the two primers remains intact following arestriction digestion. If the amount of total and intact DNA is known,the amount of cleaved DNA can be determined. Since cleavage of the DNAdepends on the methylation state of the DNA, the intact and cleaved DNArepresents different methylation states.

Amplification of a DNA locus using reactions is well known (see U.S.Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications (Innis et al., eds, 1990)). Typically, PCR is used toamplify DNA templates. However, alternative methods of amplificationhave been described and can also be employed, as long as the alternativemethods amplify intact DNA to a greater extent than the methods amplifycleaved DNA.

DNA amplified by the methods of the invention can be further evaluated,detected, cloned, sequenced, and the like, either in solution or afterbinding to a solid support, by any method usually applied to thedetection of a specific DNA sequence such as PCR, oligomer restriction(Saiki, et al., Bio/Technology 3:1008-1012 (1985)), allele-specificoligonucleotide (ASO) probe analysis (Conner, et al., Proc. Natl. Acad.Sci. USA 80:278 (1983)), oligonucleotide ligation assays (OLAs)(Landegren, et al., Science 241:1077, (1988)), and the like. Moleculartechniques for DNA analysis have been reviewed (Landegren, et al.,Science 242:229-237 (1988)).

Quantitative amplification methods (e.g., quantitative PCR orquantitative linear amplification) can be used to quantify the amount ofintact DNA within a locus flanked by amplification primers followingrestriction digestion. Methods of quantitative amplification aredisclosed in, e.g., U.S. Pat. Nos. 6,180,349; 6,033,854; and 5,972,602,as well as in, e.g., Gibson et al., Genome Research 6:995-1001 (1996);DeGraves, et al., Biotechniques 34(1):106-10, 112-5 (2003); Deiman B, etal., Mol Biotechnol. 20(2):163-79 (2002). Amplifications may bemonitored in “real time.”

In general, quantitative amplification is based on the monitoring of thesignal (e.g., fluorescence of a probe) representing copies of thetemplate in cycles of an amplification (e.g., PCR) reaction. In theinitial cycles of the PCR, a very low signal is observed because thequantity of the amplicon formed does not support a measurable signaloutput from the assay. After the initial cycles, as the amount of formedamplicon increases, the signal intensity increases to a measurable leveland reaches a plateau in later cycles when the PCR enters into anon-logarithmic phase. Through a plot of the signal intensity versus thecycle number, the specific cycle at which a measurable signal isobtained from the PCR reaction can be deduced and used to back-calculatethe quantity of the target before the start of the PCR. The number ofthe specific cycles that is determined by this method is typicallyreferred to as the cycle threshold (Ct). Exemplary methods are describedin, e.g., Heid et al. Genome Methods 6:986-94 (1996) with reference tohydrolysis probes.

One method for detection of amplification products is the 5′-3′exonuclease “hydrolysis” PCR assay (also referred to as the TaqMan™assay) (U.S. Pat. Nos. 5,210,015 and 5,487,972; Holland et al., Proc.Natl. Acad. Sci. USA 88: 7276-7280 (1991); Lee et al., Nucleic AcidsRes. 21: 3761-3766 (1993)). This assay detects the accumulation of aspecific PCR product by hybridization and cleavage of a doubly labeledfluorogenic probe (the “TaqMan™” probe) during the amplificationreaction. The fluorogenic probe consists of an oligonucleotide labeledwith both a fluorescent reporter dye and a quencher dye. During PCR,this probe is cleaved by the 5′-exonuclease activity of DNA polymeraseif, and only if, it hybridizes to the segment being amplified. Cleavageof the probe generates an increase in the fluorescence intensity of thereporter dye.

Another method of detecting amplification products that relies on theuse of energy transfer is the “beacon probe” method described by Tyagiand Kramer (Nature Biotech. 14:303-309 (1996)), which is also thesubject of U.S. Pat. Nos. 5,119,801 and 5,312,728. This method employsoligonucleotide hybridization probes that can form hairpin structures.On one end of the hybridization probe (either the 5′ or 3′ end), thereis a donor fluorophore, and on the other end, an acceptor moiety. In thecase of the Tyagi and Kramer method, this acceptor moiety is a quencher,that is, the acceptor absorbs energy released by the donor, but thendoes not itself fluoresce. Thus, when the beacon is in the openconformation, the fluorescence of the donor fluorophore is detectable,whereas when the beacon is in the hairpin (closed) conformation, thefluorescence of the donor fluorophore is quenched. When employed in PCR,the molecular beacon probe, which hybridizes to one of the strands ofthe PCR product, is in the open conformation and fluorescence isdetected, and the probes that remain unhybridized will not fluoresce(Tyagi and Kramer, Nature Biotechnol. 14: 303-306 (1996)). As a result,the amount of fluorescence will increase as the amount of PCR productincreases, and thus may be used as a measure of the progress of the PCR.Those of skill in the art will recognize that other methods ofquantitative amplification are also available.

Various other techniques for performing quantitative amplification of anucleic acid are also known. For example, some methodologies employ oneor more probe oligonucleotides that are structured such that a change influorescence is generated when the oligonucleotide(s) is hybridized to atarget nucleic acid. For example, one such method involves a dualfluorophore approach that exploits fluorescence resonance energytransfer (FRET), e.g., LightCycler™ hybridization probes, where twooligo probes anneal to the amplicon. The oligonucleotides are designedto hybridize in a head-to-tail orientation with the fluorophoresseparated at a distance that is compatible with efficient energytransfer. Other examples of labeled oligonucleotides that are structuredto emit a signal when bound to a nucleic acid or incorporated into anextension product include: Scorpions™ probes (e.g., Whitcombe et al.,Nature Biotechnology 17:804-807, 1999, and U.S. Pat. No. 6,326,145),Sunrise™ (or Amplifluor™) probes (e.g., Nazarenko et al., Nuc. AcidsRes. 25:2516-2521, 1997, and U.S. Pat. No. 6,117,635), and probes thatform a secondary structure that results in reduced signal without aquencher and that emits increased signal when hybridized to a target(e.g., Lux Probes™)

In other embodiments, intercalating agents that produce a signal whenintercalated in double stranded DNA may be used. Exemplary agentsinclude SYBR GREEN™ and SYBR GOLD™. Since these agents are nottemplate-specific, it is assumed that the signal is generated based ontemplate-specific amplification. This can be confirmed by monitoringsignal as a function of temperature because melting point of templatesequences will generally be much higher than, for example,primer-dimers, etc.

C. Hybrid Capture

In some embodiments, nucleic acid hybrid capture assays can be used todetect the presence and quantity of DNA cleaved by the restrictionenzymes. This method can be used with or without previously amplifyingthe DNA. Following the restriction digests, RNA probes whichspecifically hybridize to DNA sequences of interest are combined withthe DNA to form RNA:DNA hybrids. Antibodies that bind to RNA:DNA hybridsare then used to detect the presence of the hybrids and therefore, thepresence and amount of uncut DNA.

DNA fragments that are restricted in a window of sequence that iscomplimentary to the RNA probe hybridize less efficiently to the RNAprobe than do DNA fragments that remain intact in the window of sequencebeing monitored. The amount of hybridization allows one to quantifyintact DNA, and the quantity of DNA methylation can be inferred directlyfrom the quantity of intact DNA from different restriction enzymetreatments (i.e., methylation-sensitive and/or methylation-dependentrestriction enzyme treatments).

Methods of detecting RNA:DNA hybrids using antibodies are known in theart and are described in, e.g., Van Der Pol et al., J. Clin. Microbiol.40(10): 3558 (2002); Federschneider et al., Am. J. Obstet. Gynecol.191(3):757 (2004); Pretet et al., J. Clin. Virol. 31(2):140-7 (2004);Giovannelli et al., J. Clin. Microbiol. 42(8):3861 (2004); Masumoto etal., Gynecol. Oncol. 94(2):509-14 (2004); Nonogaki et al., Acta Cytol.48(4):514 (2004); Negri et al., Am. J. Clin. Pathol. 122(1):90 (2004);Sarian et al., Gynecol. Oncol. 94(1):181 (2004); Oliveira et al., Diagn.Cytopathol. 31(1):19 (2004); Rowe et al., Diagn. Cytopathol. 30(6):426(2004); Clavel et al., Br. J. Cancer 90(9):1803-8 (2004); Schiller etal., Am. J. Clin. Pathol. 121(4):537 (2004); Arbyn et al., J. Natl.Cancer Inst. 96(4):280 (2004); Syrjanen et al., J. Clin. Microbiol. 2004February; 42(2):505 (2004); Lin et al., J. Clin. Microbiol. 42(1):366(2004); Guyot et al., BMC Infect. Dis. 25; 3(1):23 (2003); Kim et al.,Gynecol. Oncol. 89(2):210-7 (2003); Negri et al., Am J Surg Pathol.27(2):187 (2003); Vince et al., J. Clin. Virol. Suppl 3:S109 (2002);Poljak et al., J. Clin. Virol. Suppl 3:S89 (2002). In some cases, theantibodies are labeled with a detectable label (e.g., an enzymaticlabel, an isotope, or a fluorescent label) to facilitate detection.Alternatively, the antibody:nucleic acid complex may be furthercontacted with a secondary antibody labeled with a detectable label. Fora review of suitable immunological and immunoassay procedures, see,e.g., Harlow & Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring HarborPublication, New York (1988); Basic and Clinical Immunology (Stites &Terr eds., 7^(th) ed. 1991); U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168); Methods in Cell Biology: Antibodies in CellBiology, volume 37 (Asai, ed. 1993).

Monoclonal, polyclonal antibodies, or mixtures thereof may be used tobind the RNA:DNA hybrids. Detection of RNA:DNA hybrids using monoclonalantibodies is described in, e.g., U.S. Pat. Nos. 4,732,847 and4,833,084. Detection of RNA:DNA hybrids using polyclonal antibodies isdescribed in, e.g., U.S. Pat. No. 6,686,151. The polyclonal ormonoclonal antibodies may be generated with specific binding properties.For example, monoclonal or polyclonal antibodies that specifically bindto shorter (e.g., less than 20 base pairs) or longer (e.g., more than100 base pairs) RNA:DNA hybrids may be generated. In addition,monoclonal or polyclonal antibodies may be produced that are either moreor less sensitive to mismatches within the RNA:DNA hybrid.

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with RNA:DNA hybrids are known to those of skill in theart. For example, preparation of polyclonal and monoclonal antibodies byimmunizing suitable laboratory animals (e.g., chickens, mice, rabbits,rats, goats, horses, and the like) with an appropriate immunogen (e.g.,an RNA:DNA hybrid). Such methods are described in, e.g., Coligan,Current Protocols in Immunology (1991); Harlow & Lane, supra; Goding,Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler& Milstein, Nature 256:495497 (1975).

Antibodies can also be recombinantly produced. Antibody preparation byselection of antibodies from libraries of nucleic acids encodingrecombinant antibodies packaged in phage or similar vectors is describedin, e.g., Huse et al., Science 246:1275-1281 (1989) and Ward et al.,Nature 341:544-546 (1989). In addition, antibodies can be producedrecombinantly using methods known in the art and described in, e.g.,Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989);Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); andCurrent Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

D. Generation of Control Values

Control values can represent either external values (e.g., the number ofintact loci in a second DNA sample with a known or expected number ofmethylated nucleotides or methylated restriction enzyme recognitionsequences) or internal values (e.g., a second locus in the same DNAsample or the same locus in a second DNA sample). While helpful, it isnot necessary to know how many nucleotides (i.e., the absolute value) inthe control are methylated. For example, for loci in which methylationresults in a disease state, knowledge that the locus is more methylatedthan it is in normal cells can indicate that the subject from which thesample was obtained may have the disease or be in the early stages ofdeveloping disease.

In cases where the same DNA sample includes a control locus, multiplexamplification, e.g., multiplex PCR can be used to analyze two more loci(e.g., at least one target locus and at least one control locus).

DNA samples can vary by two parameters with respect to methylation: (i)the percentage of total copies in a population that have any methylationat a specific locus, and (ii) for copies with any DNA methylation, theaverage methylation density among the copies. It is ideal, though notrequired, to use control DNAs that evaluate both of these parameters ina test sample.

Control DNAs with known methylated cytosines are produced using anynumber of DNA methylates, each of which can have a different targetmethylation recognition sequence. This procedure can create a populationof DNA fragments that vary with respect to the methylation density(i.e., the number of methylated cytosines per allele). Partial methylasereactions can also be used, e.g., to produce a normally distributedpopulation with a mode at the average methylation density for thepopulation. In some embodiments, the mode can be adjusted for a givenpopulation as a function of the completeness of the methylase reaction.Control DNAs can also be synthesized with methylated and unmethylatedDNA bases.

In some cases, a DNA target with a known sequence is used. A desiredcontrol DNA can be produced by selecting the best combination ofmethylases and restriction enzymes for the analysis. First, a map ofsites that can be methylated by each available methylase is generated.Second, a restriction map of the locus is also produced. Third,methylases are selected and are used to in vitro methylate the controlDNA sample to bring about a desired methylation pattern, which isdesigned to perform optimally in combination with the restrictionenzymes used in the methylation analysis of the test DNA and control DNAsamples. For example, M.HhaI methylates the site (G*CGC) and McrBCrecognizes two half sites with the motif (RpC). Therefore, eachmethylated M.HhaI site in the control sequence is recognized by McrBC.

Similarly, a population of molecules may be then treated with a DNAmethylase (e.g., M.SssI) in the presence of magnesium to result in adesired methylation density. If the reaction is allowed to run tocompletion, nearly all of the sites that can be methylated will bemethylated, resulting in a high and homogeneous methylation density. Ifthe reaction is limited in its course, a lower average methylationdensity (or partial methylation) will result (i.e., all possible sitesare not methylated due to timing of reaction and/or concentration ofenzyme). In this way, the desired average methylation density of thecontrol DNA can be produced. The methylated control DNA can be preciselycharacterized by determining the number of methylated cytosines throughbisulfite sequencing. Alternatively, the methylation control DNA can beprecisely characterized by determining the number of methylatedcytosines through a comparison to other known control DNAs as describedherein.

For more precise prediction of methylation densities, it may be usefulto generate a control set of DNA that can conveniently serve as astandard curve, where each sample in the control set has a differentmethylation density, either known or unknown. By cutting the multiplesamples with a methylation-dependent restriction enzyme or amethylation-sensitive restriction enzyme under conditions that allow forat least some copies of potential restriction enzyme cleavage sites inthe locus to remain uncleaved and subsequently amplifying the remainingintact copies of a locus, a standard curve of the amount of intactcopies (e.g., represented by Ct values) can be generated, therebycorrelating the amount of intact DNA to different methylation densities.The standard curve can then be used to determine the methylation densityof a test DNA sample by interpolating the amount of intact DNA in thesample following restriction and amplification as described herein.

E. Methylation State-Specific Amplification

In some embodiments, methylation-specific PCR can be employed to monitorthe methylation state of specific nucleotides in a DNA locus. In theseembodiments, following or preceding digestion with the restrictionenzyme, the DNA is combined with an agent that modifies unmethylatedcytosines. For example, sodium bisulfite is added to the DNA, therebyconverting unmethylated cytosines to uracil, leaving the methylatedcytosines intact. One or more primers are designed to distinguishbetween the methylated and unmethylated sequences that have been treatedwith sodium bisulfite. For example, primers complementary to thebisulfite-treated methylated sequence will contain guanosines, which arecomplementary to endogenous cytosines. Primers complementary to thebisulfite-treated unmethylated sequence will contain adenosine, whichare complementary to the uracil, the conversion product of unmethylatedcytosine. Preferably, nucleotides that distinguish between the convertedmethylated and unmethylated sequences will be at or near the 3′ end ofthe primers. Variations of methods using sodium bisulfite-based PCR aredescribed in, e.g., Herman et al., Proc. Natl. Acad. Sci. USA93:9821-9826 (1996); U.S. Pat. Nos. 5,786,146 and 6,200,756.

F. Detection of Methylation Associated with Disease

Amplification primers can be designed to amplify loci associated with aparticular phenotype or disease. Detection of altered methylationprofiles at loci where such alterations are associated with disease canbe used to provide diagnoses or prognoses of disease. See, e.g.,Table 1. See, also, Costello and Plass, J Med Genet 38:285-303 (2001)and Jones and Baylin, Nature. Rev 3:415-428 (2002).

TABLE 1 Examples of Genes Exhibiting Hypermethylation in Cancer Effectof loss of function in tumor Gene development Tumor types RB Loss ofcell-cycle control Retinoblastoma MLH1 Increased mutation rate, drugColon, ovarian, endometrial, gastric resistance BRCA1 Genomicinstability Breast, ovarian E-CAD Increased cell motility Breast,gastric, lung, prostate, colon, leukemia APC Aberrant cell transductionBreast, lung, colon, gastric, esophageal, pancreatic, hepatocellular p16Loss of cell-cycle control Most tumor types VHL Altered proteindegradation Clear-cell renal cell carcinoma p73 Loss of cell-cyclecontrol Leukemia, lymphoma, ovarian RASSF1A Aberrant cell transductionLung, breast, ovarian, kidney, nasopharyngeal p15 Loss of cell-cyclecontrol Leukemia, lymphoma, gastric, squamous cell carcinoma,hepatocellular GSTP1 Increased DNA damage Prostate DAPK Reducedapoptosis Lymphoma, lung MGMT Increased mutation rate Colon, lung,brain, esophageal, gastric P14ARF Loss of cell cycle control Melanoma,non-melanoma skin cancer, pancreatic, breast, head and neck, lung,mesothelioma, neurofinromatosis, colon, soft tissue sarcoma., bladder,Hodgkin's, Ewing's sarcoma, Wilm's tumor, osteosarcoma, rhabdomyosarcomaATM Defective DNA repair Leukemia, lymphoma CDKN2B Loss of cell cyclecontrol Breast, ovarian, prostate FHIT Defective DNA repair Lung,pancreas, stomach, kidney, cervix, breast MSH2 Defective DNA repairColon NF1/2 Loss of cell cycle control Neurofibroma PTCH Loss of cellcycle control Skin, basal and squamous cell carcinomas, brain PTEN Lossof cell cycle control Breast, thyroid, skin, head and neck, endometrialSMAD4 Loss of cell cycle control Pancreas, colon SMARCA3/B1 Loss of cellcycle control Colon STK11 Loss of cell cycle control Melanoma,gastrointestinal TIMP3 Disruption of cellular matrix Uterus, breast,colon, brain, kidney TP53 Loss of cell cycle control; reduced Colon,prostate, breast, gall bladder, bile duct, apoptosis BCL2 Loss of cellcycle control; reduced Lymphoma, breast apoptosis OBCAM Loss of cellcycle control Ovarian GATA4 Transcriptional silencing of Colorectal,gastric, ovary downstream genes GATA5 Transcriptional silencing ofColorectal, gastric, ovary downstream genes HIC1 Loss of cell cyclecontrol Epithelium, lymphoma, sarcoma Abbreviations: APC, adenomatouspolyposis coli; BRCA1, breast cancer 1; DAPK, death-associated proteinkinase; E-cad, epithelial cadherin; GSTP1 glutathione S-transferase π1;MLH1, MutL homologue 1, MGMT, O(6)-methylguanine-DNA methyltransferase;p15, p15^(INK4b); p16, p16^(INK4); p73, p73; Rb, retinoblastoma;RASSF1a, Ras association domain family 1A; VHL, von Hippel-Lindau; ATM,ataxia telangiectasia mutated; CDKN2, cyclin dependent kinase inhibitor;FHIT, fragile histidine triad; MSH2, mutS homologue 2; NF½,neurofibromin ½; PTCH, patched homologue; PTEN, phosphatase and tensinhomologue; SMAD4, mothers against decapentaplegic homologue 4;SMARCA3/B1, SWI/SNF-related, matrix-associated, actin-dependentregulator of chromatin, subfamily A, member 3/subfamily B, member 1;STK11, serine/threonine kinase 11; TIMP3, tissue inhibitor ofmetalloproteinase 3; Bcl-2m B-call CLL/Lymphoma 2; OBCAM, opoid-bindingcell adhesion molecule; GATA, globin transcription factor; HIC1,hypermethylated in cancer.

For example, methylation of the p16 locus is associated with pancreaticcancer. See, e.g., Schutte et al., Cancer Res. 57:3126-3131 (1997).Methylation changes at the insulin-like growth factor II/H19 locus inkidney are associated with Wilms tumorigenesis. See, e.g., Okamoto etal., Proc. Natl. Acad. Sci. USA 94:5367-5371 (1997). The association ofalteration of methylation in the p15, E-cadherin and von Hippel-Lindauloci are also associated with cancers. See, e.g., Herman et al., Proc.Natl. Acad. Sci. USA 93:9821-9826 (1997). The methylation state of GSTP1is associated with prostate cancer. See, e.g., U.S. Pat. No. 5,552,277.

Genomic DNA samples can be obtained by any means known in the art. Incases where a particular phenotype or disease is to be detected, DNAsamples should be prepared from a tissue of interest, or as appropriate,from blood. For example, DNA can be prepared from biopsy tissue todetect the methylation state of a particular locus associated withcancer. The nucleic acid-containing specimen used for detection ofmethylated loci (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)) may be from any source and may beextracted by a variety of techniques such as those described by Ausubelet al., Current Protocols in Molecular Biology (1995) or Sambrook etal., Molecular Cloning, A Laboratory Manual (3rd ed. 2001). Exemplarytissues include, e.g., brain, colon, urogenital, hematopoietic, thymus,testis, ovarian, uterine, prostate, breast, colon, lung and renaltissue.

Detection and identification of loci of altered methylation (compared tonormal cells) in DNA samples can indicate that at least some of thecells from which the sample was derived are diseased. Such diseasesinclude but are not limited to, e.g., low grade astrocytoma, anaplasticastrocytoma, glioblastoma, medulloblastoma, colon cancer, liver cancer,lung cancer, renal cancer, leukemia (e.g., acute lymphocytic leukemia,chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloidleukemia), lymphoma, breast cancer, prostate cancer, cervical cancer,endometrial cancer, neuroblastoma, cancer of the oral cavity (e.g.,tongue, mouth, pharynx), esophageal cancer, stomach cancer, cancer ofthe small intestine, rectal cancer, anal cancer, cancer of the analcanal and anorectum, cancer of the intrahepatic bile duct, gallbladdercancer, biliary cancer, pancreatic cancer, bone cancer, cancer of thejoints, skin cancer (e.g., melanoma, non-epithelial cancer, basal cellcarcinoma, squamous cell carcinoma), soft tissue cancers, uterinecancer, ovarian cancer, vulval cancer, vaginal cancer, urinary cancer,cancer of the ureter, cancer of the eye, head and neck cancer,non-Hodgkin lymphoma, Hodgkin lymphoma, multiple myeloma, brain cancer,cancer of the nervous system. Identification of altered methylationprofiles is also useful for detection and diagnosis of loss of genomicimprinting, fragile X syndrome and X-chromosome inactivation.

If desired, multiplex DNA methods can be used to amplify multipletargets from the same sample. The additional targets can representcontrols (e.g., from a locus of known methylation status) or additionalloci associated with a phenotype or disease.

In some embodiments, the methods of the invention are used to identifynew loci associated with a disease phenotype, such as cancer, or areused to validate such an association.

F. Exemplary Methods of Determining Relative Methylation at a Locus

As described above, a number of possibilities are available fordetermining the relative amount of methylation at a genetic locus ofinterest. For example, partial or complete digestions can be performed,methylation-sensitive or methylation-dependent restriction enzymes canbe used, sodium bisulfite treatment can be employed, etc. Withoutintending to limit the invention to a particular series of steps, thefollowing possibilities are further exemplified.

In some embodiments, a DNA sample is digested (partially or tocompletion) with McrBC or another methylation-dependent restrictionenzyme and a locus is subsequently amplified using quantitative DNAamplification (e.g., PCR, rolling circle amplification, and othermethods known to those skilled in the art). The resulting kineticprofiles of the amplification reactions are compared to those derivedfrom a similarly treated control DNA sample. Kinetic profiles ofamplification reactions can be obtained by numerous means known to thoseskilled in the art, which include fluorescence reaction monitoring ofTaqMan™, molecular beacons, intercalating dye (e.g., Sybr Green™)incorporation, SCORPION™ probes, and others.

In some embodiments, the DNA sample is split into equal portions and oneportion is treated with the methylation-dependent restriction enzyme andthe other is not. The two portions are amplified and compared todetermine the relative amount of methylation at the locus.

In some embodiments, the DNA sample can be split into equal portions,wherein each portion is submitted to a different amount of partialdigestion with McrBC or another methylation-dependent restrictionenzyme. The amount of intact locus in the various portions (e.g., asmeasured by quantitative DNA amplification) can be compared to a controlpopulation (either from the same sample representing uncut DNA orequivalent portions from another DNA sample). In cases where theequivalent portions are from a second DNA sample, the second sample canhave an expected or known number of methylated nucleotides (or at leastmethylated restriction enzyme recognition sequences) or, alternatively,the number of methylated recognition sequences can be unknown. In thelatter case, the control sample will often be from a sample ofbiological relevance, e.g., from a diseased or normal tissue, etc.

In some embodiments, the DNA sample is partially digested with one ormore methylation-sensitive restriction enzymes and then amplified toidentify intact loci. Controls in these cases are similar to those usedfor methylation-dependent restriction enzyme digestions described above.Untreated controls are undigested, and any treated control DNA samplesare digested with methylation-sensitive restriction enzymes.

In some embodiments, a sample is separated into at least two portions.The first portion is digested with an enzyme from one of the threepossible methylation-sensing classes of restriction enzymes (i.e.,methylation sensitive, methylation insensitive, and methylationdependent). Each additional portion is digested with the isoschizomericpartner from a different methylation-sensing class from the enzyme usedto digest the first portion. The intact loci are then amplified andquantified. The relative methylation at the locus can be determined bycomparing the results obtained from any two of the reactions to eachother, with or without comparison to an undigested portion. In the casewhere methylation insensitive enzymes are used, the portion typicallyundergoes a partial digestion.

In some embodiments, the DNA sample is treated with an agent thatmodifies unmethylated cytosine, but leaves methylated cytosineunmodified, e.g., sodium bisulfite. The sample is separated into equalportions, and one portion is treated with a methylation-dependentrestriction enzyme (e.g., McrBC). Sodium bisulfite treatment does notmodify McrBC recognition sites because sodium bisulfite modifiesunmethylated cytosine and the recognition site of each McrBC hemi-siteis a purine base followed by a methylated cytosine. Samples from bothcut and uncut portions are then amplified using at least one primer thatdistinguishes between methylated and unmethylated nucleotides. Theamplified portions are then compared to determine relative methylation.Certain quantitative amplification technologies employ one or moredetection probes that are distinct from the amplification primers. Thesedetection probes can also be designed to discriminate between convertedmethylated and unmethylated DNA. In some embodiments, the detectionprobes are used in combination with a methylation-dependent restrictionenzyme (e.g., McrBC). For example, the detection probes can be used toquantify methylation density within a locus by comparing the kineticamplification profiles between a converted McrBC treated sample and aconverted sample that was not treated with McrBC.

Alternatively, in some embodiments, the sample is divided into equalportions and one portion is digested (partially or completely) with amethylation-dependent restriction enzyme (e.g., McrBC). Both portionsare then treated with sodium bisulfite and analyzed by quantitativeamplification using a primer that distinguishes between convertedmethylated and unmethylated nucleotides. The amplification products arecompared to each other as well as a standard to determine the relativemethylation density.

In some embodiments, the DNA sample is divided into portions and oneportion is treated with one or more methylation-sensitive restrictionenzymes. The digested portion is then further subdivided and onesubdivision is digested with a methylation-dependent restriction enzyme(e.g., McrBC). The various portions and subportions are then amplifiedand compared. Following digestion, the portions and subportions canoptionally be treated with sodium bisulfite and amplified using at leastone primer that distinguishes between methylated and unmethylatednucleotides.

In some embodiments, the DNA sample is divided into four portions: afirst portion is left untreated, a second portion is contacted with amethylation-sensitive restriction enzyme (wherein intact sequences aremethylated), a third portion is contacted with a methylation-dependentrestriction enzyme (wherein intact sequences are unmethylated), and afourth portion is contacted with a methylation-sensitive restrictionenzyme and a methylation-dependent restriction enzyme in which one ofthe restriction enzymes in the fourth portion is contacted to the sampleunder conditions that allow for at least some copies of potentialrestriction enzyme cleavage sites in the locus to remain uncleaved(e.g., under partial digest conditions and/or using McrBC). See, FIG.13. If desired, a fifth portion of the sample can be analyzed followingtreatment with a methylation insensitive isoschizomer of amethylation-dependent or methylation-sensitive restriction enzyme usedin another portion, thereby controlling for incomplete digestions and/ormutations at the restriction enzyme recognition sequence. In addition todigestion, the portions and subportions can optionally be treated withsodium bisulfite and amplified using at least one primer thatdistinguishes between methylated and unmethylated nucleotides.

III. Calculation of Methylation Density Based on Cycle Thresholds

As described above, cycle thresholds (Ct) are a useful measurement fordetermining the initial amount of DNA template in an amplificationreaction. Accordingly, Ct values from samples treated with amethylation-dependent and/or methylation-sensitive restriction enzymeand amplified as described herein can be used to calculate methylationdensity at recognition sequences of the methylation-sensitive ormethylation-dependent restriction enzymes used. A change in Ct valuebetween one sample and a control value (which can represent the Ct valuefrom a second sample) is predictive of relative methylation density.Because amplification in PCR theoretically doubles copies every cycle,2^(X) approximates the number of copies in the amplification duringexponential amplification, where X is the number of cycles. Thus 2^(Ct)is proportional to the amount of intact DNA at the initiation ofamplification. The change of Ct (ΔCt) between two samples or between asample and a control value (e.g., representing a Ct value from acontrol) represents the difference in initial starting template in thesamples. Therefore, 2^(|ΔCt|) is proportional to the relativemethylation density difference between a sample and a control or asecond sample. For instance, as explained in Example 9, a difference of1.46 in the Ct between two samples (each treated with amethylation-dependent restriction enzyme and subsequently amplified)indicates that one sample has at least 2.75 (i.e., 2^((1.46))=2.75)times more potential methylated restriction sites within the locus thanthe other sample.

VI. Kits

The present invention also provides kits for performing the methods ofthe invention. For example, the kits of the invention can comprise,e.g., a methylation-dependent restriction enzyme or a methylationsensitive restriction enzyme, a control DNA molecule comprising apre-determined number of methylated nucleotides, and one or twodifferent control oligonucleotide primers that hybridize to the controlDNA molecule. In some cases, the kits comprise a plurality of DNAmolecules comprising different pre-determined numbers of methylatednucleotides to enable the user to compare amplification of a sample toseveral DNAs comprising a known number of methylated nucleotides.

The kits of the invention will often contain written instructions forusing the kits. The kits can also comprise reagents sufficient tosupport the activity of the restriction enzyme. The kits can alsoinclude a thermostable DNA polymerase.

In some cases, the kits comprise one or two different targetoligonucleotide primers that hybridize to a pre-determined region ofhuman genomic DNA. For example, as described above, the primers canallow for amplification of loci associated with the development orprognosis of disease.

In some embodiments, the kits may comprise one or moredetectably-labeled oligonucleotide probes to monitor amplification oftarget polynucleotides.

In some embodiments, the kits comprise at least one targetoligonucleotide primer that distinguishes between modified unmethylatedand methylated DNA in human genomic DNA. In these embodiments, the kitsalso typically include a fluorescent moiety that allows the kineticprofile of any amplification reaction to be acquired in real time.

In some embodiments, the kits comprise at least one targetoligonucleotide primer that distinguishes between modified unmethylatedand methylated DNA in human genomic DNA. In these embodiments, the kitswill also typically include an agent that modifies unmethylatedcytosine.

In some embodiments, the kits comprise an RNA probe, a binding agent(e.g., an antibody or an antibody mimetic) that specifically bindsRNA:DNA complexes, detection reagents, and methylation sensitive and/ormethylation dependent restriction enzymes.

EXAMPLES Example 1 Constructing a DNA Methylation Standard Sample Set

A standard sample set is generated in numerous ways. For example, amethylase (e.g., M.SssI or other methylates such as MHhaI, M.AluI) isapplied in vitro to a series of DNA samples to produce a standard set ofDNAs known to have increasing methylation densities. This standard setis generated by first obtaining a sample of known sequence (e.g., thelocus of interest). Next, the sample is divided into a series of samplesand each sample in the series is treated with the chosen methylase inthe presence of magnesium and in a manner that results in increasingmethylation densities of the samples in the series.

A partial methylation reaction refers to contacting DNA with a cocktailof one or more methylases under appropriate reaction conditions suchthat the methylase modifies some (e.g., about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%) but not all of the possible methylase recognitionsites for each enzyme in the methylase cocktail. A DNA sequence ispartially methylated by treating DNA with an active methylase for ashorter period of time than is necessary to achieve completemethylation, and then terminating the reaction, or under other alteredreaction conditions that allow for the desired amount of partialmethylation.

The methylation densities of each sample in the series are measured bysequencing a statistically significant sample of clones from abisulfite-treated portion of each series member in the set, byidentifying the converted cytosines within each clone, and bycalculating the average methylation density for each reaction within themethylation sample set. In order to achieve a partial methylationdensity on a given fragment, the methylase acts in a stochastic manner,and not a processive manner. For M.SssI, this is achieved by conductingthe reaction in the presence of magnesium, since M.SssI methylates DNAin a processive way in the absence of magnesium, while in the presenceof magnesium, the enzyme methylates CpGs in a non processive, stochasticmanner.

Example 2 Quantitatively Determining the Relative Methylation of a Locusof Interest Between One Tissue and Another Tissue with QuantitativeAmplification

DNA is collected from two sources: a test population (diseased) and acontrol population (normal).

Each population of DNA fragments is similarly submitted to variouspartial or complete digestions with the enzyme McrBC. McrBC recognizestwo R^(M)C sites, each a half site, that are within 40 to 3,000 basesand with an optimal separation of the half sites of 50-103 bp and thencuts the DNA fragment sometimes 3′ of both half sites, sometimes 3′ ofthe 5′ most half site and 5′ of the 3′ most half sites, and sometimes 5′of both half sites.

Next, the digested DNA in each population is amplified and the amount ofthe amplified locus is measured as a function of cycle number. Thegreater the number of methylated half sites in the locus of interest ona given DNA fragment within the population studied, the greater theprobability that McrBC will cut between the PCR primers, and, therefore,a greater number of amplification cycles will be required to achieve theidentical concentration of amplified PCR locus.

To determine whether the locus of interest within the test population ismore or less methylated than the locus of interest within the controlpopulation, a concentration curve of amplified DNA of the testpopulation is compared to the concentration curve of amplified DNA fromthe control population. Concentration curves reflect the amount ofintact DNA as a function of the amount of digestion in a series ofdifferent partial digestions.

Example 3 Measuring the Methylation Density at a Locus of Interestwithin a Tissue with Quantitative Amplification

DNA is obtained from a single source, and is divided into twopopulations. The first population of DNA is completely digested with theenzyme McrBC, while the remaining population is untreated.Alternatively, the first population is digested with a cocktail of oneor more methylation sensitive restriction enzymes (e.g., HpaII, HhaI, orAciI, etc.), while the second population of DNA is untreated.

Next, the digested DNA in the first population is amplified and theamount of the amplified locus is measured as a function of cycle number.The greater the number of methylated half sites in the locus of intereston a given DNA fragment within the population studied, the greater theprobability that McrBC will cut between the PCR primers, and, therefore,a greater number of amplification cycles will be required to achieve theidentical concentration of amplified PCR locus. Alternatively, when acocktail of methylation sensitive restriction enzymes is used, thegreater the number of methylated restriction sites in the locus ofinterest on a given DNA fragment within the population studied, thelower the probability that the methylation sensitive cocktail of enzymeswill cut between the PCR primers. Therefore, a lower number ofamplification cycles will be required to achieve the identicalconcentration of amplified PCR locus.

To determine whether the locus of interest within the first populationis methylated, a comparison is made between the kinetics of theamplification reaction profiles from the treated and untreatedpopulations. Alternatively, to determine the density of methylationwithin the tissue at the locus of interest, the kinetics of theamplification reaction profiles are compared to those obtained from aknown in vitro generated methylation sample set, i.e., a standardmethylation curve.

Example 4 Measuring the Methylation Density at a Locus of Interestwithin a Tissue with Amplification End Point Analysis

DNA is obtained from a single source, and is divided into a series oftwo or more portions.

This series is exposed to an increasing amount of partial digestion by amethylation dependent restriction enzyme, such as McrBC. The firstportion of DNA fragments is untreated, the second portion is lightlydigested with McrBC, and subsequent populations are more fully digested(but less than completely) with McrBC. The range of partial digestionsis obtained through the manipulation of reaction conditions, such as thetitration of enzyme amounts, digestion times, temperatures, reactants,buffers, or other required components.

Next, the DNA from the series of portions are amplified and the amountof amplified PCR loci is measured after a fixed number of cycles. Thegreater the number of methylated half sites in the locus of interest ona given DNA fragment within the first McrBC-treated portion, the greaterthe probability that McrBC will cut fragments of the first part betweenthe PCR primers, and the greater number of amplification cycles will berequired to detect a certain concentration of amplified PCR locus in thefirst portion.

To determine whether the locus of interest within the test population ismore or less methylated, the results obtained from the series ofportions and the parallel analysis of the standard sample set arecompared (see Example 1).

Example 5 Quantifying Methylation Using Methylation-SensingIsoschizomeric Partners and Quantitative PCR

DNA is collected from two sources: a test population (diseased) and acontrol population (normal). Each population is divided into groups oftwo or more portions.

Each group is exposed to an increasing amount of partial digestion by amethylation sensitive restriction enzyme (e.g., HpaII, MboI (A)). Thefirst portion of DNA fragments is untreated, the second portion islightly digested with the methylation sensitive restriction enzyme, andsubsequent populations are more fully digested (but less than tocompletion) with the enzyme. The range of partial digestions is obtainedthrough the manipulation of reaction conditions, such as the titrationof enzyme amounts, digestion times, temperatures, reactants, buffers, orother required components.

The second group of portions is similarly digested with anisoschizomeric partner of a different methylation-sensing class from theenzyme used to treat the first group of portions (e.g., MspI and Sau3AI(A), respectively). Alternatively, the second group of portions remainsuntreated.

Next, all of the portions in the groups are amplified and the kineticreaction profile from each amplification is obtained. Alternatively, endpoint analysis after a fixed number of cycles is used.

To determine whether the locus of interest within the test population ismore or less methylated, a comparison is made between the kineticreaction profiles between the groups (group vs. group). Additionally, todetermine whether the locus of interest between the two tissues is moreor less methylated, a comparison is made between the kinetic reactionprofiles between the populations (diseased groups vs. normal groups).

Example 6 Quantifying the Methylation Density of a Locus of InterestUsing a Cocktail of Methylation Sensitive Enzymes

DNA is obtained from a single source and is divided into groups of twoor more portions. Alternatively, DNA is collected from two sources: atest population (diseased) and a control population (normal), and isdivided into groups of two or more uniform portions.

The groups of uniform portions are treated with a fixed number of unitsof a cocktail of one of more methylation sensitive restriction enzymes(e.g., HpaII, HaeIII) for varied amounts of time.

Next, all of the portions in the groups are amplified and the kineticreaction profile from each amplification is obtained. Alternatively, endpoint analysis after a fixed number of cycles is used.

To determine whether the locus of interest within the test population ismore or less methylated, a comparison is made between the kineticreaction profiles between the groups (group vs. group). Additionally, todetermine whether the locus of interest between the two tissues is moreor less methylated, a comparison is made between the kinetic reactionprofiles between the populations (diseased groups vs. normal groups).Finally, the overall amount of methylation can be determined bycomparing these results to those obtained from the standard sample set(see Example 1).

Example 7 Quantifying the Methylation Density of a Small Population ofMethylated Alleles in the Presence of a Large Population of UnmethylatedAlleles

DNA is obtained from a single source and is divided into two portions.Alternatively, DNA is collected from two sources: a test population(diseased) and a control population (normal), and each population isdivided into two portions.

To discriminate between methylated and unmethylated alleles, one portionfrom each population is treated with sodium bisulfite, which convertsthe unmethylated cytosine residues to uracil, leaving unconvertedmethylated cytosine residues. The bisulfite-treated portion is dividedinto two equal subportions. Alternatively, one portion from eachpopulation is digested with a cocktail of one or more methylationsensitive restriction enzymes (e.g., HpaII, HhaI, etc.), leaving theremaining portion untreated. The digested portion is similarly dividedinto two equal subportions.

One of the bisulfite-treated subportions is completely digested with theenzyme McrBC, while the remaining subportion is untreated.Alternatively, one of the methylation sensitive restrictionenzyme-treated subportions is completely digested with the enzyme McrBC,while the remaining subportion is untreated.

One or both of the amplification primers are designed to resemble thebisulfite converted sequence overlapping at least one methylatedcytosine residue. In this way, only those fragments that belong to thesubset of fragments that were methylated at that primer in the testpopulation have the potential of becoming amplified, while thosefragments in the subset of fragments that remained unmethylated in thelocus of interest will not be amplified. Alternatively, if methylationsensitive enzymes are used to discriminate between methylated andunmethylated alleles, then primers designed to the native sequence areused and only alleles that were methylated at the recognition sitesremain intact and will be amplified.

Next, the DNA from both the McrBC-treated and McrBC-untreated portions,along with the relevant controls, are amplified and the amount ofamplified PCR loci are measured as a function of cycle number.

To determine whether the locus of interest within the first populationis methylated, a comparison is made between the kinetics of theamplification reaction profiles from the treated and untreatedpopulations. To determine the density of methylation within the tissueat the locus of interest, the kinetics of the amplification reactionprofiles are compared to those obtained from a known in vitro generatedmethylation sample set, i.e., a standard methylation curve.

Alternatively, this Example could also be performed by reversing theorder of the sodium bisulfite conversion and the McrBC-digestion stepsdescribed above (i.e., McrBC digestion takes place prior to sodiumbisulfite conversion).

In another alternative, partial digestion using McrBC is used in eithera subportion or a series of subportions, instead of complete digestion.

Example 8 Demonstrating the Sensitivity of Detection

Human male placental DNA was obtained and was methylated in vitro usingM.SssI, which methylates cytosines (5 mC) when the cytosines arefollowed by guanosine (i.e., GC motifs). The resulting in vitromethylated DNA was then mixed into unmethylated male placental DNA atknown ratios, thereby producing a set of mixes, each comprising adifferent percentage of total copies that are methylated.

The various mixtures were then divided into three portions: an uncutportion; a portion digested with HhaI, a methylation-sensitiverestriction enzyme that is sensitive to 5 mC and having the recognitionsequence GCGC, where underlined nucleotides are unmethylated; and aportion digested with both HhaI and McrBC. McrBC is amethylation-dependent restriction enzyme that cleaves in the proximityof its methylated recognition sequence. The digested sequences weresubsequently amplified using primers specific for a region upstream ofthe CDKN2A (p16) gene in the human genome [Ensembl geneID#ENSG00000147889]. This region was determined to be unmethylated inhuman male placental DNA that has not been methylated in vitro. Theprimer sequences were:

Forward primer (SEQ ID NO: 2) 5′-CGGGGACGCGAGCAGCACCAGAAT-3′,Reverse primer (SEQ ID NO: 3) 5′CGCCCCCACCCCCACCACCAT-3′and standard PCR conditions were used:

-   -   1 cycle [at 95° C. for 3 minutes]    -   followed by 49 cycles at [95° C. for 30 sec, 65° C. for 15        seconds, and 68° C. for 15 seconds, a plate read (68° C.) and        then another plate read at 83° C.].

The second plate reading at 83° C. was conducted to eliminate thefluorescence contribution of primer dimers to the reaction profile. Amelt-curve, which measures fluorescence as a function of temperature,was performed between 80° C. and 95° C. at the end of the cycles andproduct specificity was determined. The locus of interest is 181 bp inlength and has a melting temperature of approximately 89° C.Amplification product accumulation was determined using theintercalating dye, SYBR Green™ Dynamo Kit from MJ Research, whichfluoresces when it binds to double stranded nucleic acids, and reactionswere cycled and fluorescent intensity was monitored using the MJ OpticonII Real-time PCR machine.

A threshold at which the signal from the amplification products could bedetected above background was determined empirically from a parallelanalysis of a template dilution standard curve. The threshold wasadjusted to maximize the fit of the regression curve (Ct vs. log [DNA]),according to standard threshold determination protocols familiar tothose skilled in the art, such as those described in e.g., Fortin etal., Anal. Biochem. 289: 281-288 (2001). Once set, the threshold wasfixed and the cycle thresholds (Ct) for each reaction were calculated bythe software (MJ Research Opticon II Monitor V2.02). As expected, thederived cycle thresholds increased at higher dilutions of methylated tounmethylated DNA (FIG. 1). Also shown in FIG. 1, the change (or “shift”)in cycle threshold (ΔCt) between uncut DNA and the HhaI treated DNAcorresponded with that expected (E) for the dilutions, demonstratingthat cycle threshold shift can be used to accurately predict therelative proportion of copies that are methylated in the sample out ofthe total number of copies in the sample.

FIG. 1 also illustrates that the addition of HhaI (a methylationsensitive restriction enzyme) and McrBC (a methylation-dependentrestriction enzyme) further alters the Ct compared to the samplestreated with HhaI alone. The degradation in the number of intact copies,and the resultant Ct shift to a higher Ct value after treatment with themethylation dependent restriction enzyme and the methylation sensitiveenzyme further confirms the assessment that the intact copies presentafter treatment with the methylation-sensitive restriction enzyme aloneare in fact methylated. In other words, this double digest provides acontrol against the possibility that HhaI was not added, was inactive,was partially active, or otherwise did not result in a complete digest.The addition of the methylation-dependent restriction enzyme and itsability to destroy methylated templates confirms the results observedafter treatment with just the methylation-sensitive restriction enzymeand provides an internal control to assess the completeness of themethylation-sensitive restriction enzyme reaction.

FIG. 2 depicts the kinetic profile of four portions at three dilutionsof methylated DNA to unmethylated DNA. In each of the three dilutions,all four portions were digested first with the methylation sensitiverestriction enzyme HhaI. The first two portions in each dilution weredigested with McrBC, and the second two portions in each dilution wereuntreated with regard to McrBC. All portions were then amplified underidentical conditions and the fluorescence intensities were measured.Three observations can be made. First, the duplicate reactions havenearly identical Ct values, demonstrating that the assays are highlyreproducible. Second, decreasing change in Ct between treated anduntreated portions as a function of increasing dilution of themethylated copies shows that as the methylated gene copies get morerare, there is less difference between the Ct values observed betweenthe McrBC treated and untreated portions. This suggests that the HhaIand HhaI+McrBC reactions will converge and that at some point we willnot be able to monitor methylation density or be able to identify thepresence or absence of methylated copies. A theoretical extinction ofdetection will occur at a ΔCt of zero. Using a regression analysis, wesolved for the extinction function in our system and found that thedilution where delta Ct=0 is 1:20,000, methylated copies to unmethylatedcopies respectively. This regression analysis is detailed in FIG. 4.

FIG. 2 shows the fluorescent kinetic profile of a series of portions alldiluted to 1:2,000 methylated copies to unmethylated copiesrespectively. While the overall fluorescence obtained from the 1:2,000reactions is not ideal, one can see a difference between the HhaI andthe HhaI/McrBC reactions. Notice that the McrBC digestion destroys theaccumulation of the fluorescence, and the melting point curve in FIG. 3shows a specific peak at 89° C., which is the predicted meltingtemperature for the 181 bp specific amplicon. Here we are clearlydetecting merely 1.4 cellular equivalents of methylated DNA diluted intoa total of 2,762 cellular equivalents of DNA.

As shown in FIG. 4, 1.4 cellular equivalents (CE) were detected out of atotal of 2,764 CE in the tube having a total of 20 ng of genomic DNA.Each cellular equivalent has approximately 7.9 pg of genomics DNA percell. Thus, if 50 ng of genomic DNA is used, one methylated copy in thepresence of 10,000 unmethylated copies should be detectable. Thisprinciple is illustrated in FIG. 4. FIG. 5 provides a breakdown of thisanalysis. Note that this detection limit can be further lowered by (i)using an optimized FRET-based probe, rather than an intercalating dye,to detect amplified products, (ii) by further optimizing PCR primerdesign, or (iii) by further optimizing PCR reaction conditions.

Example 9 Detecting Methylation Density

This example demonstrates determining the average density of methylation(i.e., the average number of methylated nucleotides) within a locus. Asprovided in FIG. 6, it is likely that in many diseases, methylation ofone or more loci goes through a progression of increased methylationdensity corresponding to disease progression. Previously-describedmethylation detection techniques involve detecting the presence orabsence of methylation at one or more particular nucleotides but do notprovide analysis of density across a locus. In contrast, the presentinvention provides methods for detecting the average number ofmethylation events within a locus.

As illustrated in FIGS. 11-12, methods that detect methylation at onlyspecific short sequences (typically relying on primer or probehybridization) may miss changes in methylation (see FIG. 11) that thepresent methylation density detection methods (which examine relativemethylation across an entire locus) are able to detect (see FIG. 12).

This discovery works by treating a locus with a methylation-dependent ormethylation-sensitive restriction enzyme under conditions such that atleast some copies of potential restriction enzyme cleavage sites in thelocus to remain uncleaved. While these conditions can be achieved byallowing for partial digestion of a sample, the particular recognitionand cutting activity of McrBC allows for additional options.

As discussed above, when two McrBC complexes meet, restriction occurs,typically within ˜32 bp of either half site (i.e., in one of fourregions). See FIG. 7. Restriction does not occur if half sites arecloser than 20 bp.

Since McrBC randomly cuts 5′ or 3′ of the recognized pair of half sites,the probability of cutting at a locus (spanned by primers in the casePCR) is function of the number of methylated half-sites present at ornear the locus. For a set concentration of enzyme and time ofincubation, the more methylation sites within a locus, the greater theprobability McrBC will cut at the locus (or between the primers in thecase of PCR). However, under ideal circumstances and sufficient numberof DNA copies, the probability that McrBC will cut every copy of a locusis low because it will sometimes cut at a distance outside of the locus,thereby leaving the locus intact. Thus, the number of intact loci isinversely proportional to the average number of methylated nucleotideswithin the locus. The number of intact loci is inversely proportional tothe Ct value for a given sample. Thus, the Ct value is proportional tothe average number of methylated nucleotides within a locus. Thus,comparison of the Ct value of amplified McrBC-treated DNA compared tothe Ct value from amplified untreated DNA allows for the determinationof methylation density of the locus.

Two aliquots of BAC DNA containing the p16 locus was in vitro methylatedat different densities. The first aliquot was densely methylated withM.sssI. There are 20 M.sssI methylase sites within the PCR amplicon, 11of which are also McrBC half-sites. The second aliquot was sparselymethylated with M.HhaI. There are four M.HhaI methylase sites within thePCR amplicon, all four of which are also McrBC half-sites. Within thePCR amplicon there are also 4 restriction sites for HhaI. All four ofthese HhaI restriction sites are methylase sites for both M.sssI andM.HhaI, such that complete treatment with either methylase will protectall four HhaI sites from restriction. A different number of units ofMcrBC was used for a set period of time (four hours) to generate aseries of progressively more partial digestions to identify an amount ofenzyme to best allow for distinguishing results from the sparsely anddensely-methylated DNA. As displayed in FIGS. 8 and 9, the Ct valueswere proportional to the concentration of McrBC used in both sparselyand densely-methylated sequences. FIG. 10 demonstrates results fromtitrating different amounts of McrBC to enhance resolution betweensparsely and densely methylated sequences to distinguish between thetwo. In FIG. 10, “1×” equals 0.8 units of McrBC as defined by NewEngland Biolabs.

The densely methylated target has 2.75-fold more methylated McrBC halfsites than the sparsely methylated target (11/4=2.75). Therefore, upontreatment with McrBC and subsequent amplification, we expect to see adifference between the Ct of the reactions of about 1.46, as2^(ΔCt)=2.75. Solving for ΔCt, ΔCt=log(2.75)/log(2)=1.46. We observedΔCt (sparse−dense @ 1× McrBC) was 1.51±0.05. Thus, the methylationdensity of a locus was determined using this method.

Example 10 Bisulfite-Coupled Methylation Density Determination

This example demonstrates the ability to determine the methylationdensity of a locus by treatment with both bisulfite and a methylationdependent restriction enzyme followed by PCR amplification andquantitation of the amplified products.

Two samples of DNA, one purified from human blood cells and the otherpurified from a glioma cell line, were treated with bisulfite. Thesamples were then each split into two portions, one portion from eachwas digested with McrBC, while the other portion was mock-digested (i.e.was not digested with McrBC). Since methylation (5 mC) is protected frombisulfite conversion, all McrBC sites remain intact in the convertedDNA.

From each of the four portions, 1 μL, 2.5 μL and 5 μL, respectively, wasutilized as template for PCR amplification, resulting in 12 PCRreactions. A no template negative control and a bisulfite treatedpositive control were also analyzed. PCR primers, which were designed toanneal to the bisulfite converted sequence of a locus of interest, andPCR reagents were used in the 12 PCR reactions and in the positive andnegative control reactions. PCR amplification of the locus was conductedfor a number of cycles determined to be limiting and equal volumealiquots of the amplifications were evaluated with agarose gelelectrophoresis.

The lanes labeled “untreated” in the agarose gel image in FIG. 14represent bisulfite converted DNA from glioma (left) and blood (right)that were not digested with McrBC. The lanes labeled “McrBC” in theagarose gel image in FIG. 14 represent bisulfite converted DNA fromglioma (left) and blood (right) that were digested with McrBC. McrBCtreatment resulted in a decrease in PCR amplicon signal from bothsamples, suggesting that both samples contain at least some 5 mC.Additionally, PCR amplicon signal of the McrBC treated blood aliquotswas greater than the PCR amplicon signal of the McrBC treated gliomaaliquots, suggesting that the density of McrBC in the glioma sample wasgreater than the density of McrBC in the blood sample.

To independently determine the density of methylation in the samples,bisulfite sequencing was performed on approximately ten and thirtycloned PCR amplicons from the bisulfite treated glioma and bloodsamples, respectively. Sequence analysis was conducted to tabulate thepercentage of methylation at each CpG in the locus of interest for eachof the samples. CpG positions in the locus are indicated as tick in thetop line in FIG. 14, and the second row of graphs in FIG. 14 depictsmethylation density of each CpG in each sample. The bars (red in theglioma graph and green in the blood graph) illustrate the percentage oftimes that each CpG was sequenced as methylated (i.e., it was sequencedas “C” rather than “T” following bisulfite treatment, amplification andcloning and sequencing). The absolute methylation density determined bybisulfite sequencing was 92% in Glioma cells and 7% in normal bloodcells. The independent confirmation and the McrBC-coupled bisulfite PCRresults above agreed.

Example 11 Methylation Density Determination

This example demonstrates the ability of methylation-dependent andmethylation-sensitive restriction enzymes to distinguish differentmethylation densities at a locus.

A 703 bp portion of the promoter of p16 was amplified. The portion wasmethylated in vitro in a time course with M.SssI under conditions thatpromote stochastic methylation. The portion is illustrated in FIG. 15.From the large methylation reaction at different time points, fixedvolumes (20 μl) were removed and the methylation reactions were stoppedwith heat (65° C.). We stopped one reaction before it began (T=1 is 0minutes methylation, i.e., the unmethylated control; T=2 was stopped at2 minutes; T=3 was stopped at 5 minutes; and T=4 was stopped at 60minutes, a time at which the PCR product in the reaction should havebeen fully methylated.

The reactions were purified and each amplicon then was diluted more than1 million fold in TE buffer, and was added back to the human genome at aratio that should approximate a normal copy balance (i.e., two copiesper 7.9 picograms). The human genome used was homozygous for a deletionof the p16 gene. The deletion cell line is CRL-2610. This allowed us toadd a fixed amount of the human genome (i.e., control for the complexityof the genome in our reaction).

DNA samples were cleaved with Aci I (a methylation-sensitive restrictionenzyme), McrBC (a methylation-dependent restriction enzyme), or both asdouble digest, and the portion was amplified. Amplicons were detectedwith the MS_p16(207) SYBR green real-time PCR system. Twenty nanogramsof input DNA (genome+amplicon) equal ˜2764 cellular equivalents/per PCRreaction. Each set of four digests was brought up to volume inrestriction salts with BSA and GTP such that it could be split into fourtubes (˜4 μg). Each of the four digest tubes (˜1 μg) had 100 μl totalvolume such that 2 μl could be added to PCR reactions, thereby adding 20ng of DNA. Digests were allowed to proceed for four hours and were heatkilled for 20 minutes. PCR conditions:

(SEQ ID NO: 4) CAGGGCGTCGCCAGGAGGAGGTCTGTGATT = F primer (SEQ ID NO: 5)GGCGCTGCCCAACGCACCGAATAGTTACGG = R primer

Dynamo MJ qPCR buffer, 65° C. anneal, two cycle PCR (95° C. 30 sec, 65°C. 20 sec) cycled 49 times and monitored with an MJ opticon IIquantitative PCR system.

We hypothesized that if the technology is monitoring density:

a) The McrBC cleavage should demonstrate a larger ΔCT for each sample ina progression from 0 ΔCt for T=1, up to a maximal ΔCt at T=4 (60minutes)

b) The Aci I reactions should demonstrate the inverse relationship.

c) The mock treated and double digests should be fixed reference points

As illustrated in FIG. 16, we observed the trends outlined above. TheMcrBC curve moves oppositely from the Aci I curve, and the movement isin proportion with the increasing methylation content in the locus. Theuntreated and double digests indicate the boundaries of the assay field.The system resolves the difference between each of the reactions alongthe time course, such that each graphical depiction showing the varioustimed reactions is different. The point where the profile intersects thedashed threshold line indicates the point where information is compared.

Another way to visualize the data is by plotting the change in cyclethreshold values (ΔCt). See FIG. 17. FIG. 17 displays the ΔCt forMcrBC-treated compared to untreated at each time point in the partialmethylation reaction, and the corresponding ΔCt for the Aci I digests(Aci I compared to untreated). As expected, the McrBC and Aci I ΔCtlines provide an intersecting inverse pattern. The Aci I graph displaysa chunky shape because its cut sites are fixed, while McrBC displays asmooth continuous distribution, reflecting its ability to cut more orless randomly following site recognition. Frequency of cutting isproportional to the expected change in methylation occupancy based uponthe time course. The error bars associated with the real-timemeasurements are indicated. If they are not visualized, they are withinthe data point.

Example 12 Monitoring DNA Methylation of a Target Sequence Present atMultiple Locations in the Genome

This example demonstrates the ability of the present technology todetermine the methylation of a target sequence that is present in agenome more than one time (i.e., more than one copy) using an assay thatmonitors a sequence repeated in the kafirin gene cluster in Sorgumbicolor.

Eleven kafirin genes were annotated from the publicly available sequenceof a BAC clone AF527808 from Sorghum bicolor. PCR primers were designedto amplify a 247 bp amplicon form all 11 kafirin genes (the primersequences were conserved in all 11).

The forward primer was (SEQ ID NO: 6) 5′ CTCCTTGCGCTCCTTGCTCTTTC 3′The reverse primer was (SEQ ID NO: 7) 5′ GCTGCGCTGCGATGGTCTGT 3′

Sorghum genomic DNA isolated from seedling leaf was divided into 6 equalportions. The six portions were treated in the following manner: i)untreated (mock treated), ii) HhaI digested, iii) McrBC digested, iv)HhaI and McrBC digested, v) PstI digested and, vi) PstI and McrBCdigested. Equal volume aliquots from the six portions were amplifiedusing the above PCR primers in the following manner:

The SYBR green real-time PCR cycling parameters were 95° C. for 3minutes, followed by 50 cycles of 2 step PCR 95° C. for 30 sec, 56° C.for 30 seconds with the Dynamo Kit from MJ Research (Boston, Mass.). Weutilized both a low temperature (70° C.) and a high-temperature plateread (82° C.). The input of genomic DNA was 10 ng per PCR reaction. Thethreshold was set using a template dilution standard control.

The kinetic profiles for the 6 PCR reactions are depicted in FIG. 19.The inset in FIG. 19 depicts the template dilution standard curve usedto set the cycle threshold for the experiment. Each set of 6 digests wasperformed three times, and each of the 18 digests had four PCRreplicates. The PCR reactions were determined to be highly reproducible.In FIG. 19, PCR amplification reaction kinetics for each of the sixdigestions are depicted with different colors: Red=mock treated,Blue=McrBC digested, Orange=HhaI digested, and Green=HhaI+McrBC doubledigest, Pink=PstI, and Azure=PstI+McrBC double-digest. Comparisonsbetween the cycle thresholds of the six amplified digestions were madeand of the density of CNG and CG methylation in the repeated targetsequence was determined.

In all 11 kafirin genes, all PstI sites in the repeated target sequencewere methylated (at CNG) and PstI digestion was blocked since the PstItreated sample (pink) has the same cycle threshold (Ct) as the mocktreated sample (red). This result is supported by the McrBC digestedsample (blue), which has a significantly higher Ct than themock-digested DNA control (red), further demonstrating that CNGmethylation exists because McrBC was able to cut, thereby lowering thenumber of intact copies of the target sequence. All, or almost all, ofthe PstI sites are methylated because the double PstI+McrBC digest(light blue) has the same Ct as McrBC alone (blue). Note that the McrBCdigestion with and without PstI yields the same Ct, while HhaI withMcrBC (green) yields a higher Ct on average; suggesting that not allHhaI sites were methylated and that HhaI was able to reduce the numberof intact copies of the target sequence. These results indicate thatevery target sequence has high CNG methylation covering all PstI sites,while some but not all HhaI sites are methylated, indicating partial CGmethylation of HhaI sites in the target sequence. The specificity ofeach reaction was confirmed using melt-curve analysis.

For the kafirin genes, the average difference in Ct between the McrBCsingle and HhaI+McrBC double digests is 2.46 cycles (22.08±0.34HhaI+McrBC−19.62±0.19 McrBC). We compared the cycle-thresholds ofgenomic DNA that had been subjected to various treatments and inferredmethylation occupancy through the changes in Ct mediated by thetreatments. The Ct of any locus is a function of the number of copiespresent within the assay tube. Each of the eleven genes was broken into˜1.5 kb pieces which were aligned to create a consensus kafirin assembly(FIG. 18). The consensus kafirin sequence was examined and PCR primersamplifying a 247 bp amplicon were selected (see above).

As for CG methylation, the HhaI digested (orange) sample has the same Ctas the mock treated control (red); however, the HhaI+McrBC double digest(green) has a Ct that is 2.46 cycles greater than the McrBC alone(blue), indicating that some HhaI sites must not be modified. A cyclethreshold difference of 2.46 indicates that there is 2^(2.46), orapproximately 5.5-fold, less DNA in the HhaI+McrBC double digestedsample. This suggests that 2 out of the 11 kafirin genes have someunmethylated HhaI sites.

To independently confirm the presence of methylation at the repeatedtarget sequence, a 1× shotgun sequence was generated of the methylfiltered sorghum genome (See U.S. Patent Publication No. 20010046669,Bedell et al., PLOS in press). 95% of the genes in the sorghum genomewere determined to be represented in the methyl filtered sequence set.In the kafirin gene cluster, however, only 2 of 11 genes from BAC cloneAF527808 were represented in the methyl filtered sequence set,suggesting that most or all of them may be methylated, and therefore areunderrepresented in the methyl filtered sequence. Ten of the genes aretandemly arrayed in a cluster and share an average of 99.1% sequenceidentity, while the eleventh gene is located 45 kb away and is morediverged (76.2% identity on average). A 247 bp region was selected forPCR close to the 5′end because of its near identity across all 11 genesand because of the high CG and CNG content (see FIG. 18). Theindependent confirmation of methylation at the target sequence agreedwith the methylation determination made by analysis of the reactionkinetics of the amplified digested DNA.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, databases, and patents cited hereinare hereby incorporated by reference.

1-16. (canceled)
 17. A method of calculating the relative methylationdensity for a target locus in a DNA sample, the method comprising, i.contacting a first portion of the DNA sample with amethylation-dependent restriction enzyme under partial digestionconditions where the resulting number of remaining intact copies of thelocus is inversely proportional to the quantity of methylation at thelocus; ii. quantitatively amplifying intact copies of the locus in theDNA sample after step i; iii. identifying a cycle-threshold (Ct) valuefrom step ii; iv. quantitatively amplifying intact copies of the locusin a second portion of the genomic DNA that has not been cut by arestriction enzyme; v. identifying a cycle-threshold (Ct) value fromstep iv; and, vi. determining the relative methylation density for thetarget locus by calculating the difference (ΔCt) between the Ct fromstep iii and the Ct from step v, wherein 2^(|ΔCt|) is proportional tothe relative methylation density.
 18. A method for detecting methylationdensity in a locus of genomic DNA, the method comprising: a. contactinga first portion of the genomic DNA containing the locus with amethylation-dependent restriction enzyme under partial digestionconditions where the resulting number of remaining intact copies of thelocus is inversely proportional to the quantity of methylation at thelocus; b. quantifying the remaining intact copies of the locus in thefirst portion following step a; c. quantifying intact copies of thelocus in a second portion of the genomic DNA that has not been cut by arestriction enzyme, wherein comparison of the quantity of intact copiesof the locus in step b to the quantity of intact copies of the locus instep c allows for methylation density detection.
 19. The method of claim18, further comprising: contacting a third portion of the genomic DNAwith a methylation-sensitive restriction enzyme and themethylation-dependent restriction enzyme to generate intact and cleavedcopies of the locus; quantifying the intact copies of the locus in thethird portion following methylation-sensitive restriction enzyme andmethylation-dependent restriction enzyme digestion.
 20. The method ofclaim 19, further comprising: contacting a fourth portion of the genomicDNA with a methylation-sensitive restriction enzyme to generate intactand cleaved copies of the locus; quantifying the intact copies of thelocus in the fourth portion following methylation-sensitive restrictionenzyme digestion.
 21. The method of claim 18, further comprising:contacting a third portion of the genomic DNA with amethylation-sensitive restriction enzyme to generate intact and cleavedcopies of the locus; quantifying the intact copies of the locus in thethird portion following methylation-sensitive restriction enzymedigestion.
 22. The method of claim 18, wherein the quantifying stepscomprise hybridizing two oligonucleotide primers to genomic DNA flankingthe locus to produce an amplification product corresponding to theintact copies of the locus of genomic DNA between the primers.
 23. Themethod of claim 22, wherein the quantifying steps comprise quantitativeamplification of the intact copies of the locus.
 24. The method of claim19, wherein the quantifying steps comprise quantitative amplification ofthe intact copies of the locus.
 25. The method of claim 20, wherein thequantifying steps comprise quantitative amplification of the intactcopies of the locus.
 26. The method of claim 21, wherein the quantifyingsteps comprise quantitative amplification of the intact copies of thelocus.
 27. The method of claim 23, wherein the quantitativeamplification is real-time quantitative polymerase chain reaction (PCR).28. The method of claim 23, wherein the method further comprisescontacting the genomic DNA with an agent that modifies unmethylatedcytosine before the amplification step, and at least one of the twooligonucleotide primers distinguishes between modified unmethylated andmethylated DNA in the genomic DNA.
 29. The method of claim 28, whereinthe agent is sodium bisulfite.
 30. The method of claim 18, wherein thequantifying steps comprise detecting a probe that hybridizes to thelocus.
 31. The method of claim 30, wherein the probe comprises adetectable fluorescent moiety.
 32. The method of claim 18, wherein thegenomic DNA is from an animal.
 33. The method of claim 32, wherein theanimal is a human.
 34. The method of claim 18, wherein the genomic DNAis from an organism selected from the group consisting of plants, fungiand bacteria.
 35. The method of claim 18, wherein the genomic DNA isfrom a tissue selected from the group consisting of brain tissue, colontissue, urogenital tissue, lung tissue, renal tissue, breast tissue,thymus tissue, testis tissue, ovarian tissue, and uterine tissue.